Cd33 binding agents

ABSTRACT

The present invention relates to immunotherapies that are based on myeloid cell depletion. In particular, the present invention relates to CD33 binding agents for use in such therapies, e.g. in the treatment of myeloid cell malignancies and myelodysplastic syndrome (MDS).

RELATED APPLICATIONS

This application is a continuation of co-pending U.S. application Ser.No. 15/375,313, filed Dec. 12, 2016, which is a divisional of U.S.application Ser. No. 14/742,836, filed Jun. 18, 2015, now U.S. Pat. No.9,550,833, which is a divisional of U.S. application Ser. No.13/246,022, filed Sep. 27, 2011, now U.S. Pat. No. 9,079,958, whichclaims priority to European application no. 10186468.4, filed Oct. 4,2010, the contents of which are hereby incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to immunotherapies that are based onmyeloid cell depletion. In particular, the present invention relates toCD33 binding agents for use in such therapies, e.g. in the treatment ofmyeloid cell malignancies and myelodysplastic syndrome (MDS).

BACKGROUND OF THE INVENTION

In the early 1980s CD33 was identified as a marker of myeloid leukemias(Andrews et al., Blood 62, 24-132, 1983). CD33 is a cell-surface antigenspecifically expressed on myeloid cells including myeloid leukemiacells. It is the smallest member of the siglec (sialic acid-bindingIg-related lectins) family CD33 is expressed on early multilineagehematopoietic progenitor cells and myelomonocytic precursors. It isabsent from pluripotent hematopoietic stem cells (Andrews et al.,Journal of Experimental Medicine 169, 1721-1731, 1989). It isdownregulated on mature granulocytes but retained on macrophages,monocytes and dendritic cells (Andrews et al., Blood 62, 24-132, 1983).Besides myelomonocytic cells, CD33 has also been found to be expressedon human mast cells and blood basophils (Valent et al., Blood 15;73(7):1778-85, 1989,). Monoclonal antibodies directed against CD33 areused in diagnosis of leukemia as well as for therapeutic targeting andin vitro purging of bone marrow for autologous transplantation in acutemyeloid leukemia (AML) (Duzkale et al., Biol Blood Marrow Transplant.9(6):364-72, 2003). Initial efforts in therapeutic targeting focused onthe development of immunotoxins using an anti-CD33 antibody conjugatedto the toxin ricin. Since CD33 rapidly internalizes upon antibodybinding (Audran et al., J Immunol Methods. 188(1):147-54, 1995) theimmunotoxin approach was obvious.

CD33 is a 67 KD transmembrane glycoprotein. The sialic acid-bindingextracellular domain of CD33 is involved in cell-cell adhesion. Theintracellular immunoreceptor tyrosine-based inhibitory motifs (ITIM)confer inhibitory signals to the cell, affecting proliferation and cellsurvival. The actual signaling pathways of CD33 are poorly understoodbut are assumed to involve the ITIM and ITIM-like motifs and therecruitment of tyrosine phosphatases (von Gunten et al., Ann. N.Y. Acad.Sci. 1143: 61-82, 2008). A murine CD33 orthologue has been defined butits functional comparability to human CD33 was questioned (Brinkman-Vander Linden et al., Mol Cell Biol., 23(12): 4199-206, 2003). Thefunctional role of human CD33 on normal and malignant leukocytes remainsunknown.

Several publications have described CD33 as a stable cell surface markeron primary AML and CML cells expressed by 70-100% of tested patients(Plesa et al., Cancer 112(3), 572-80, 2007, Hauswirt et al., Eur J ClinInvest. Jan 73-82, 2007, Scheinberg et al., Leukemia Vol. 3, 440-445,1989). CD33 is expressed on malignant myeloid blast cells, whichrepresent the majority of malignant cells in peripheral blood and bonemarrow of leukemia patients, and on leukemic stem cells, a relativelysmall number of less differentiated cells in the bone marrow which arecharacterized by their capacity for self-renewal and the maintenance ofthe leukemic clonal hierarchy. Depletion of leukemic stem cells isregarded the key mechanism for sustained tumor free survival. The CD33targeting immunotoxin Mylotarg®, a humanized IgG₄ antibody conjugated tothe toxin chaliceamicin is used for the treatment of AML patients bydelivering its toxic payload to CD33 positive AML cells (Amadori et al.,Cancer Treat Rev. 34(1):49-60, 2008). Lintuzumab (SGN-33, HuM195), a“naked” CD33 specific humanized monoclonal antibody was evaluated inphase II clinical trials for the treatment of AML and MDS with initialclinical signs of efficacy from a phase I dose escalation study andtolerable adverse events being reported (Raza et al. Abstract #983,14^(th) EHA Congress, Jun. 4-7, 2009).

Targeting AML cell lines with CD33 specific HuM195 in vitro reducesTNF-α induced secretion of inflammatory cytokines like IL-8, MCP-1 andRANTES (Sutherland et al., Mabs 1:5, 481-490, 2009). The relevance ofthis effect for AML therapy is unknown but modulating the cytokinemilieu of the tumor microenvironment may contribute to the therapeuticefficacy of the antibody. In addition, the antibody inducesantibody-dependent cell-mediated cytotoxicity (ADCC) andantibody-dependent cell-mediated phagocytosis (ADCP) of AML cell linesin vitro (Sutherland et al., Mabs 1:5, 481-490, 2009). ADCC isconsidered to be a decisive mechanism for anti-tumor activity ofantibodies in hematological malignancies. Data from clinical trials withthe CD20-specific monoclonal antibody Rituximab have demonstrated thesignificance of effector cell mediated mechanisms for treatment ofB-cell malignancies with respect to response to antibody treatment (Wengand Levy, J Clin Oncol. 21 (21): 3940-7, 2003).

In conclusion, it has been shown that CD33 antigen is expressed onnormal cells of the myelomonocytic lineage and frequently expressed ontumor cells in myeloid leukemias. In a phase I trial with an antibodyagainst CD33 (lintuzumab) first signs of efficacy were observed withoutsevere adverse events. However, clinical development of lintuzumab wasdiscontinued after results from a phase II trial in combination withchemotherapy did not yield the expected improvement in efficacy.Therefore, there is a clear need for the development of improvedCD33-targeting treatment modalities.

In view of the prior art there is a need for providing a furtherimproved therapy for myeloid cell malignancies and MDS, particularly foracute myeloid leukemia.

In particular there is a need for providing further improvedantagonistic binding agents to CD33 for treating cancer, in particularAML.

SUMMARY OF THE INVENTION

The present invention provides novel CD33 binding agents binding tohuman CD33 and that are defined by

-   -   a) having a heavy chain variable region comprising CDR1, CDR2        and CDR3, and a light chain variable region comprising CDR4,        CDR5 and CDR6, wherein CDR1 has an amino acid sequence selected        from Seq ID No:29-42, CDR2 has an amino acid sequence selected        from Seq ID No:14-28, CDR3 has an amino acid sequence selected        from Seq ID No:1-14, CDR4 has an amino acid sequence selected        from Seq ID No:71-84, CDR5 has an amino acid sequence selected        from Seq ID No:57-70, CDR6 has an amino acid sequence selected        from Seq ID No:43-56, or    -   b) recognizing an epitope within the amino acid sequence        FFHPIPYYDKNSPVHGYW (Seq ID No: 141) of human CD33.

The present invention further provides CD33 binding agents wherein theinternalization kinetics of the CD33 binding agents are such that atleast 30%, preferably 40%, of the initial amount of the antibody remainon the cell surface of HL60 cells at a time of 4 hours after incubation.

The present invention further provides CD33 binding agents, wherein theheavy chain variable region comprises an amino acid sequence selectedfrom Seq ID No:85-98 and the light chain variable region comprises anamino acid sequence selected from Seq ID No:99-112.

The present invention further provides CD33 binding agents, wherein theheavy chain having an amino acid sequence selected from Seq IDNo:113-126 and the light chain having an amino acid sequence selectedfrom Seq ID No: 127-140.

The present invention further provides CD33 binding agents havingmutations in the F_(c) domain that increase ADCC.

Further preferred embodiments are outlined in the followingspecification and in the claims.

The CD33 binding agents according to the present invention have beenfound to have high affinity to human CD33 and further to have favourableinternalization kinetics, which is characterized by a long presence ofthe CD33 binding agents when bound to CD33 on the surface of the targetcells, which translates into favourable ADCC activity.

The inventors have also found that the CD33 binding agents according tothe present invention bind to a different epitope of the extracellulardomain of CD33 compared to Lintuzumab. Without wishing to be bound byany particular theory this is believed to be the reason for thedifferent internalization kinetics of the CD33 binding agents accordingto the present invention and Lintuzumab.

DESCRIPTION OF THE FIGURES

FIGS. 1-3 show the internalization of exemplary CD33 binding agentsaccording to the invention in comparison to lintuzumab on HL60 cells.

FIG. 4 shows the internalization rate on HL60 cells of two exemplaryantibodies according to the invention in comparison to lintuzumab.

FIG. 5 shows the ADCC performance on HL60 cells of two exemplaryantibodies according to the invention in comparison to lintuzumab.

DETAILED DESCRIPTION OF THE INVENTION

CD33 Binding Agents

The term “binding agent” as used herein means a protein or peptide thatspecifically binds to a target antigen. A binding agent can be, forexample, an antibody, a derivative of such an antibody, or other agentthat specifically binds to the target antigen. A binding agent can alsobe a protein comprising an Fv region or a portion thereof (e.g., a V_(H)or V_(L) or a CDR(s) of an antibody that specifically binds to thetarget antigen). In a preferred embodiment herein the binding agent isan antibody.

The term “CD33 binding agent” as used herein refers to a binding agentthat specifically binds to CD33, typically a portion of theextracellular domain of human CD33.

The term “antibody” as used herein refers to (a) immunoglobulinpolypeptides and immunologically active portions of immunoglobulinpolypeptides (i.e., polypeptides of the immunoglobulin family, orfragments thereof, that contain an antigen binding site thatimmunospecifically binds to a specific target antigen), or (b)conservatively substituted derivatives of such immunoglobulinpolypeptides or fragments that immunospecifically bind to the targetantigen. Antibodies are generally described in, for example, Harlow andLane, Antibodies: A Laboratory Manual (Cold Spring Harbor LaboratoryPress, 1988). The term “antibody” refers to intact monoclonalantibodies, polyclonal antibodies, monospecific antibodies,multispecific antibodies (e.g., bispecific antibodies), and antibodyfragments that exhibit the desired biological activity (e.g.,antigen-binding). The antibody can be of any type or class (e.g., IgG,IgE, IgM, IgD, and IgA) or sub-class (e.g., IgG1, lgG2, IgG3, IgG4, IgA1and IgA2), preferably of the IgG class, more preferably an IgG1.

An “intact” antibody is one which comprises an antigen-binding variableregion as well as a light chain constant domain and heavy chain constantdomains as appropriate for the antibody class. The constant domains maybe native sequence constant domains (e.g., human native sequenceconstant domains) or amino acid sequence variants thereof.

An “antibody fragment” comprises a portion of an antibody, including theantigen-binding or variable region or a portion thereof. Examples ofantibody fragments include Fab, Fab′, F(ab′)₂, and Fv fragments, V_(H)and V_(L) antigen binding fragments, diabodies, triabodies, tetrabodies,single-chain antibody, scFv, scFv-Fc, a SMTP, and multispecificantibodies formed from antibody fragment(s).

“Heavy chain variable region” or “V_(H)” means the part of the heavychain comprising the CDR1, CDR2 and CDR3 and surrounding frameworkregions.

“Light chain variable region” or “V_(L)” means the part of the lightchain comprising the CDR4, CDR5 and CDR6 and surrounding frameworkregions.

“CDR” means the hypervariable regions of the heavy and light chains,which determine the complementarity/binding specifity of an antibody orantibody fragment. The order of the CDRs in the present application ispurely numerically.

“Epitope” herein means a part of an antigen, which is recognized by anantibody or antibody fragment. In particular this term refers to partsof CD33, which can be recognized by an antibody.

“mAbs” as used herein refers to monoclonal antibodies.

An antibody may have one or more “effector functions” which refer tothose biological activities attributable to the Fc region (a nativesequence Fc region or amino acid sequence variant Fc region) of anantibody. Examples of antibody effector functions include CIq binding;complement dependent cytotoxicity (CDC); Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor; BCR), etc.

“Single-chain Fv” or “scFv” antibody fragments comprise the V_(H) andV_(L) domains of an antibody, wherein these domains are present in asingle polypeptide chain. The Fv polypeptide typically further comprisesa polypeptide linker between the V_(H) and V_(L). domains which enablesthe scFv to form the desired structure for antigen-binding. For a reviewof scFv, see Plückthun in The Pharmacology of Monoclonal Antibodies,vol. 113, Rosenburg and Moore, eds., Springer-Verlag, New York, pp.269-315 (1994).

A binding agent such as an antibody that is “directed to,” “which binds”or that “specifically binds” an antigen of interest (i.e., a targetantigen) is one capable of binding that antigen with sufficient affinitysuch that the binding agent is useful in targeting a cell expressing theantigen. Typically, the binding agent binds with an affinity of at leastabout 1×10⁷ M⁻¹, and binds to the predetermined antigen with an affinitythat is at least two-fold greater than its affinity for binding to anon-specific antigen (e.g., BSA, casein) other than the predeterminedantigen or a closely-related antigen.

An “antibody derivative” as used herein refers to an antibody, asdefined above, that is modified by covalent attachment of a heterologousmolecule such as, e.g., by attachment of a heterologous polypeptide, orby glycosylation, deglycosylation, acetylation or phosphorylation orother modification not normally associated with the antibody. In someembodiments, the heterologous molecule is not a therapeutic agent. Insome embodiments, the heterologous molecule does not exhibit acytostatic or cytotoxic effect by itself.

A further comprehensive reference for all terms and procedures usedherein is Sambrook at al., Molecular Cloning, Cold Spring HarborLaboratory Press; 3^(rd) edition (Jan. 15, 2001).

The CD33 binding agents specifically bind to a receptor, CD33,associated with a given target cell population. CD33 is a member of thesialoadhesion family that is expressed on cells of the hematopoieticlineage, including myeloid precursors, monocytes, macrophages, dendriticcells, and mast cells. CD33 is also expressed on tumor cells associatedwith myeloproliferative or mast cell proliferative diseases, includingacute myeloid leukemia and myeloplastic syndromes, and on leukemic stemcells. Antibodies targeting CD33 and their uses have been generallydescribed (see, e.g., Pierelli et al., 1993, Br. J. Haematol. 84:24-30;Matutes et al., 1985, Hemaiol. Oncol. 3: 179-186; Taussig et al., 2005,Blood 106:4086-4092; Florian et al., 2006, Leuk. & Lymph. 47:207-222).

In some embodiments, the CD33 binding agent is an antibody (e.g., amonoclonal antibody). Useful monoclonal antibodies can be homogeneouspopulations of antibodies to a CD33 (e.g., the extracellular domain ofhuman CD33). A monoclonal antibody (mAb) can be prepared by using anytechnique known in the art. These include, but are not limited to thehybridoma technique originally described by Köhler and Milstein (1975,Nature 256:495-497), the human B cell hybridoma technique (Kozbor etal., 1983, Immunology Today 4:72), and the EBV-hybridoma technique (Coleet al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc., pp. 77-96). Such antibodies may be of any immunoglobulin classincluding IgG, IgM, IgE, IgA and IgD and any subclass thereof. Thehybridoma producing a monoclonal antibody may be cultivated in vitro orin vivo.

Useful monoclonal antibodies include, but are not limited to, humanmonoclonal antibodies, humanized monoclonal antibodies, chimericmonoclonal antibodies and functionally active antibody fragments of anyof these.

Useful CD33 antibodies include antibodies that can achieve a therapeuticeffect by various mechanisms known in the art, such asantibody-dependent cell-mediated cyotoxicity (ADCC), anti-dependent cellphagocytosis (ADCP) and/or complement dependent cytotoxicity (CDC). Forexample the antibody can mediate ADCC by interacting with immuneeffector cells such as NK cells, monocytes, and macrophages.

Recombinant antibodies, such as chimeric and humanized monoclonalantibodies, comprise both human and non-human portions, which can bemade using standard recombinant DNA techniques. (See, e.g., Cabilly etal., U.S. Pat. No. 4,816,567; and Boss et al., U.S. Pat. No. 4,816,397;both of which are incorporated herein by reference in their entirety).“Humanized antibodies” are antibody molecules from non-human specieshaving one or more complementarity determining regions (CDRs) from thenon-human species and a framework region from a human immunoglobulinmolecule. (See, e.g., Queen, U.S. Pat. No. 5,585,089, which isincorporated herein by reference in its entirety.) Such chimeric andhumanized monoclonal antibodies can be produced by recombinant DNAtechniques known in the art, for example using methods described inInternational Publication No. WO 87/02671; European Patent PublicationNo. 0184187; European Patent Publication No. 0171496; European PatentPublication No. 0173494; International Publication No. WO 86/01533; U.S.Pat. No. 4,816,567; European Patent Publication No. 012023; Berter etal., 1988, Science 240: 1041-1043; Liu et al., 1987, Proc. Natl. AcadSet. USA 84:3439-3443; Liu et al., 1987, J. Immunol. 139:3521-3526; Sunet al., 1987, Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al.,1987, Cancer. Res. 47:999-1005; Wood et al. 1985, Nature 314:446-449;Shaw et al, 1988, J. Natl. Cancer Inst. 80: 1553-1559; Morrison, 1985,Science 229: 1202-1207; Oi et al., 1986, BioTechniques 4:214; U.S. Pat.No. 5,225,539; Jones et al., 1986, Nature 321:552-525; Verhoeyan et al.,1988, Science 239:1534; and Beidler et al., 1988, J. Immunol.141:4053-4060: each of which is incorporated herein by reference in itsentirety.

Human monoclonal antibodies may be made by any of numerous techniquesknown in the art (see, e.g., Teng et al., 1983, Proc. Nail. Acad. Sci.USA. 80:7308-7312; Kozbor et al, 1983, Immunology Today 4:72-79; Olssonat al., 1982, Meth. Enzymol. 92:3-16; and U.S. Pat. Nos. 5,939,598 and5,770,429).

Fully human antibodies can be produced using transgenic mice that areincapable of expressing endogenous immunoglobulin heavy and light chainsgenes, but which can express human heavy and light chain genes. Thetransgenic mice are immunized in the normal fashion with a selectedantigen, e.g., all or a portion of a CD33 polypeptide. Monoclonalantibodies directed against the antigen can be obtained usingconventional hybridoma technology. The human immunoglobulin transgenesharbored by the transgenic mice rearrange during B cell differentiation,and subsequently undergo class switching and somatic mutation. Thus,using such a technique, it is possible to produce therapeutically usefulIgG, IgA, IgM and IgE antibodies. For an overview of this technology forproducing human antibodies, see, e.g., Lonberg and Huszar (1995, Int.Rev. Immunol. 13:65-93). For a detailed discussion of this technologyfor producing human antibodies and human monoclonal antibodies andprotocols for producing such antibodies, see, e.g., U.S. Pat. Nos.5,625,126; 5,633,425; 5,569,825; 5,661,016; and 5,545,806. Other humanantibodies can be obtained commercially from, for example, Medarex(Princeton, N.J.), which are obtained by immunizing mice.

Fully human antibodies that recognize a selected epitope also can begenerated using a technique referred to as “guided selection”. In thisapproach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a fully human antibodyrecognizing the same epitope. (See, e.g., Jespers et al, 1994,Biotechnology 12:899-903). Human antibodies also can be produced usingvarious techniques known in the art, including phage display libraries(see, e.g., Hoogenboom and Winter, 1991, J. MoI. Biol 227:381; Marks etal, 1991, J MoI Biol 222:581; Quan and Carter, 2002, The rise ofmonoclonal antibodies as therapeutics, In Anti-IgE and Allergic Disease,Jardieu and Fick Jr., eds., Marcel Dekker, New York, N.Y., Chapter 20.pp. 427-469).

Useful antibody fragments include, but are not limited to, F(ab′)₂fragments, Fab′ fragments, Fab fragments, Fvs, single chain antibodies(SCAs) (e.g., as described in U.S. Pat. No. 4,946,778: Bird, 1988,Science 242:423-42; Huston et al., 1988, Proc. Natl. Acad. Sci. USA85:5879-5883; and Ward et al, 1989, Nature 334:544-54), scFv, scFv-Fc,FvdsFv, minibodies, diabodies, triabodies, tetrabodies, SMIPs (see,e.g., Published U.S. Patent Application No. 2005-0238646) and any othermolecule comprising one or more CDRs and that has the same specificityas the antibody.

In other embodiments, the antibody is a fusion protein of an antibody,or a functionally active fragment thereof, joined to another protein.For example, an antibody or antibody fragment can be fused via acovalent bond (e.g., a peptide bond), at either the N-terminus or theC-terminus to an amino acid sequence of another protein (or portionthereof, typically at least a 10, 20 or 50 amino acid portion of theprotein) that is not the antibody or an antibody fragment. In someembodiments, the antibody or fragment thereof can be covalently linkedto the other protein at the C-terminus of the variable domain or aconstant domain.

Antibodies can be modified, e.g., by the covalent attachment of any typeof molecule as long as such covalent attachment permits the antibody toretain its antigen-binding immunospecificity. For example, a derivativeof an antibody can be one that has been further modified, e.g., byglycosylation. de-glycosylation, acetylation, pegylation,phosphorylation, amidation. derealization by known protecting/blockinggroups, proteolytic cleavage, linkage to another protein, etc. Any ofnumerous chemical modifications can be carried out by known techniques,including, but not limited to. specific chemical cleavage, acetylation,formylation, metabolic synthesis in the presence of tunicamycin, or thelike. Additionally, the derivative can contain one or more unnaturalamino acids.

In specific embodiments, it may be desirable to improve the bindingaffinity and/or other biological properties of the antibody. (See, e.g.,U.S. Patent Publication Nos. 2006-0003412 and 2006-0008882) Amino acidsequence variants of the antibodies are prepared by introducingappropriate nucleotide changes into the antibody nucleic acid, or bypeptide synthesis. Such modifications include, for example, deletionsfrom, and/or insertions into and/or substitutions of, residues withinthe amino acid sequences of the antibody. Any combination of deletions,insertions, and/or substitutions is made to arrive at the finalconstruct, provided that the final construct possesses the desiredcharacteristics. The amino acid changes also may alterpost-translational processes of the antibody, such as changing thenumber or position of glycosylation sites.

A useful method for identification of certain residues or regions of theantibody that are favored locations for mutagenesis is called “alaninescanning mutagenesis” as described by Cunningham and Wells (1989,Science 244: 1081-1085). Here, a residue or group of target residues areidentified (e.g., charged residues such as arg, asp, his. lys, and glu)and replaced by a neutral or negatively charged amino acid (typicallyalanine or polyalanine) to affect the interaction of the amino acidswith antigen. Those amino acid locations demonstrating functionalsensitivity to the substitutions then are refined by introducing furtheror other variants at, or for, the sites of substitution. Thus, while thesite for introducing an amino acid sequence variation is predetermined,the nature of the mutation per se need not be predetermined. Forexample, to analyze the performance of a mutation at a given site,alanine scanning or random mutagenesis is conducted at the target codonor region and the expressed antibody variants are screened for thedesired activity.

Amino acid sequence insertions can include amino- and/orcarboxyl-terminal fusions ranging in length from one residue topolypeptides containing a hundred or more residues, as well asintrasequence insertions of single or multiple amino acid residues.Examples of terminal insertions include an antibody with an N-terminalmethionyl residue or the antibody fused to a cytotoxic polypeptide.

Another type of antibody is an amino acid substitution variant of anantibody. Such variants have at least one amino acid residue in theantibody molecule replaced by a different residue. The sites of greatestinterest for substitutional mutagenesis include the hypervariableregions, but framework region alterations are also contemplated.

Substantial modifications in the biological properties of the antibodycan be accomplished by selecting substitutions that differ significantlyin their effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain.Naturally-occurring residues are divided into groups based on commonside-chain properties:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophilic: cys, ser, thr;

(3) acidic: asp, glu;

(4) basic: asn, gin, his, lys. arg;

(5) residues that influence chain orientation: gly, pro; and

(6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

It is desirable to modify the antibody with respect to effectorfunction, e.g., so as to enhance antibody-dependent cell-mediatedcyotoxicity (ADCC), anti-dependent cell phagocytosis (ADCP) and/orcomplement dependent cytotoxicity (CDC) of the antibody. In particular,the ADCC activity can be enhanced by introducing amino acid mutations inthe constant region of the antibody (Lazar et al., PNAS 103, 11,4005-4010, 2006). This may be achieved by introducing one or more aminoacid substitutions in an Fc region of the antibody, see, e.g., PublishedU.S. Patent Application No. 2006-0160996. Alternatively or additionally,cysteine residue(s) may be introduced in the Fc region, thereby allowinginterchain disulfide bond formation in this region. The homodimericantibody thus generated may have improved internalization capabilityand/or increased CDC and ADCC. (See. e.g., Caron et al., 1992, J Exp.Med 176:1191-1195; and Shopes, 1992, J. Immunol. 148:2918-2922.)Homodimeric antibodies with enhanced anti-tumor activity may also beprepared using heterobifunctional cross-linkers as described in Wolff etal., 1993, Cancer Research 53:2560-2565. Alternatively, an antibody canbe engineered which has dual Fc regions and may thereby have enhancedcomplement lysis and ADCC capabilities. (See. e.g., Stevenson et al.,1989, Anti-Cancer Drug Design 3:219-230.)

A variety of modifications of the Fc region have been suggested in theart, both in the scientific literature and in patent documents, e.g. inEP 0307434, WO 9304173, WO 9734631, WO 9744362, WO 9805787, WO 9943713,WO 9951642, WO 9958572, WO 02060919, WO 03074679, WO 2004016750, WO2004029207,

WO 2004063351, WO 2004074455, WO 2004035752, WO 2004099249, WO2005077981, WO 2005092925, WO 2006019447, WO 2006031994, WO 2006047350,WO 2006053301, WO 2006088494 and WO 2007041635.

In preferred embodiments, the antibodies of the invention are Fcvariants with amino acid substitutions at positions 332 and/or 239and/or 236. In preferred embodiments, the antibodies of the inventionhave mutations in the Fc domain selected from the group of

-   -   i) a single substitution at position 332, preferably I332E;    -   ii) a combination of substitutions at positions 239 and 332,        preferably S239D/I332E;    -   iii) a combination of substitutions at positions 236 and 332,        preferably G236A/I332E;    -   iv) a combination of substitutions at positions 236, 239 and,        332, preferably G236A/S239D/I332E.

In this context it is particularly preferred to introduce mutations inthe F_(c) domain at one or more positions selected from amino acids atpositions 332 and/or 239 and/or 236 according to the Kabat EU numberingindex. Particular preferred are substitutions at positions 239 and 332,especially S239D/I332E.

The Fc variants in the antibodies of the present invention are definedaccording to the amino acid modifications that compose them. Thus, forexample, I332E is an Fc variant with the substitution I332E relative tothe parent Fc polypeptide. Likewise, S239D/I332E defines an Fc variantwith the substitutions S239D and I332E and S239D/I332E/G236A defines anFc variant with the substitutions S239D, I332E, and G236A relative tothe parent Fc polypeptide.

To increase the serum half life of the antibody, one may incorporate asalvage receptor binding epitope into the antibody (especially anantibody fragment) as described in U.S. Pat. No. 5,739,277, for example.As used herein, the term “salvage receptor binding epitope” refers to anepitope of the Fc region of an IgG molecule (e.g., IgG₁, IgG₂, IgG₃, orIgG₄) that is responsible for increasing the in vivo serum half-life ofthe IgG molecule.

Antibodies may be glycosylated at conserved positions in their constantregions (see, e.g., Jefferis and Lund, 1997, Chem. Immunol. 65:11 1-128:Wright and Morrison, 1997, TibTECH 15:26-32). The oligosaccharide sidechains of the immunoglobulins can affect the protein's function (see,e.g., Boyd et al., 1996, MoI. Immunol. 32: 1311-1318; Wittwe and Howard,1990, Biochem. 29:4175-4180), and the intramolecular interaction betweenportions of the glycoprotein which can affect the conformation andpresented three-dimensional surface of the glycoprotein (see, e.g.,Jefferis and Lund, supra; Wyss and Wagner, 1996, Current Opin. Biotech.7:409-416). Oligosaccharides may also serve to target a givenglycoprotein to certain molecules based upon specific recognitionstructures. For example, it has been reported that in a galactosylatedIgG, the oligosaccharide moiety ‘flips’ out of the inter-C(_(H)2 spaceand terminal N-acetylglucosamine residues become available to bindmannose binding protein (see, e.g., Malhotra et al. 1995, Nature Med.1:237-243). Removal by glycopeptidase of the oligosaccharides fromCAMPATH-1H (a recombinant humanized murine monoclonal IgG1 antibodywhich recognizes the CDw52 antigen of human lymphocytes) produced inChinese Hamster Ovary (CHO) cells resulted in a complete reduction incomplement mediated lysis (CMCL or CDC) (Boyd et al., 1996, MoI.Immunol. 32:131, 1-1318), while selective removal of sialic acidresidues using neuraminidase resulted in no loss of CMCL. Glycosylationof antibodies has also been reported to affect ADCC. In particular, CHOcells with tetracycline-regulated expression ofβ(1.4)-N-acetylglucosaminyltransferase III (GnTIII), aglycosyltransferase catalyzing formation of bisecting GIcNAc, wasreported to have improved ADCC activity (see, e.g., Umana et al, 1999,Nature Biotech. 17: 176-180).

Glycosylation of antibodies is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalaciosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Glycosylation variants of antibodies are variants in which theglycosylation pattern of an antibody is altered. By altering is meantdeleting one or more carbohydrate moieties found in the antibody, addingone or more carbohydrate moieties to the antibody, changing thecomposition of glycosylation (i.e., glycosylation pattern), the extentof glycosylation, or the like.

Addition of glycosylation sites to the antibody is convenientlyaccomplished by altering the amino acid sequence such that it containsone or more of the above-described tripeptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thesequence of the original antibody (for O-linked glycosylation sites).Similarly, removal of glycosylation sites can be accomplished by aminoacid alteration within the native glycosylation sites of the antibody.

The amino acid sequence is usually altered by altering the underlyingnucleic acid sequence. These methods include, but are not limited to,isolation from a natural source (in the case of naturally-occurringamino acid sequence variants) or preparation by oligonucleotide-mediated(or site-directed) mutagenesis, PCR mutagenesis, or cassette mutagenesisof an earlier prepared variant or a non-variant version of the antibody.

The glycosylation (including glycosylation pattern) of antibodies mayalso be altered without altering the amino acid sequence or theunderlying nucleotide sequence. Glycosylation largely depends on thehost cell used to express the antibody. Since the cell type used forexpression of recombinant glycoproteins, e.g., antibodies, as potentialtherapeutics is rarely the native cell, significant variations in theglycosylation pattern of the antibodies can be expected. (See, e.g., Hseet al., 1997, Biol. Chem. 272:9062-9070.) In addition to the choice ofhost cells, factors which affect glycosylation during recombinantproduction of antibodies include growth mode, media formulation, culturedensity, oxygenation, pH, purification schemes, and the like. Variousmethods have been proposed to alter the glycosylation pattern achievedin a particular host organism, including introducing or overexpressingcertain enzymes involved in oligosaccharide production (see, e.g., U.S.Pat. Nos. 5,047,335; 5,510,261; and 5,278,299). Glycosylation, orcertain types of glycosylation, can be enzymatically removed from theglycoprotein, for example using endoglycosidase H (Endo H). In addition,the recombinant host cell can be genetically engineered, e.g., madedefective in processing certain types of polysaccharides. These andsimilar techniques are well known in the art.

The glycosylation structure of antibodies can be readily analyzed byconventional techniques of carbohydrate analysis, including lectinchromatography, NMR, mass spectrometry, HPLC, GPC, monosaccharidecompositional analysis, sequential enzymatic digestion, and HPAEC-PAD,which uses high pH anion exchange chromatography to separateoligosaccharides based on charge. Methods for releasing oligosaccharidesfor analytical purposes are also known, and include, without limitation.en2ymatic treatment (commonly performed using peptide-N-glycosidaseF/endo-β-galactosidase), elimination using harsh alkaline environment torelease mainly O-linked structures, and chemical methods using anhydroushydrazine to release both N- and O-linked oligosaccharides.

The antibodies also can have modifications (e.g., substitutions,deletions or additions) in amino acid residues that interact with Fcreceptors. In particular, antibodies can have modifications in aminoacid residues identified as involved in the interaction between theanti-Fc domain and the FcRn receptor (see, e.g., InternationalPublication No. WO 97/34631).

In its broadest aspect the present invention relates to CD33 bindingagents binding to human CD33 and that are defined by

-   -   a) having a heavy chain variable region comprising CDR1, CDR2        and CDR3, and a light chain variable region comprising CDR4,        CDR5 and CDR6, wherein CDR1 has an amino acid sequence selected        from Seq ID No:29-42, CDR2 has an amino acid sequence selected        from Seq ID No:15-28, CDR3 has an amino acid sequence selected        from Seq ID No:1-14, CDR4 has an amino acid sequence selected        from Seq ID No:71-84, CDR5 has an amino acid sequence selected        from Seq ID No:57-70, CDR6 has an amino acid sequence selected        from Seq ID No:43-56, or    -   b) recognizing an epitope within the amino acid sequence        FFHPIPYYDKNSPVHGYW (Seq ID No: 141) of human CD33.

The present invention further provides CD33 binding agents wherein theinternalization kinetics of the CD33 binding agents are such that atleast 30% of the initial amount of the antibody remain on the cellsurface of HL60 cells at a time of 4 hours after incubation.

It has been found that the CD33 binding agents according to the presentinvention bind to a different epitope than lintuzumab, see example 4herein. Both epitopes (Seq ID No: 141 and Seq ID No: 142) arenon-overlapping. It is believed that the different epitopes on theexatracellular domain of CD33, which are recognized by the CD33 bindingagents according to the present invention and by lintuzumab, are thereason for the different internalization behaviour and ADCC performanceof the CD33 binding agents according to the present invention andlintuzumab (cf. examples 2 and 3 herein).

In a preferred embodiment at least 40% of the initial amount of the CD33binding agents remain on the cell surface at a time of 4 hours afterincubation.

In a preferred embodiment the heavy chain variable region comprises anamino acid sequence selected from Seq ID No:85-98 and the light chainvariable region comprises an amino acid sequence selected from Seq IDNo:99-112.

In a preferred embodiment the CD33 binding agent has a heavy chainhaving an amino acid sequence selected from Seq ID No:113-126 and alight chain having an amino acid sequence selected from Seq IDNo:127-140.

In a preferred embodiment the CD33 binding agent has an affinity to bothhuman CD33 and cynomolgus CD33 with a KD of equal to or less than 10 nM.

In a preferred embodiment the CD33 binding agent is humanized

In a preferred embodiment the CD33 binding agent is fully human.

In a preferred embodiment the CD33 binding agent further comprises aneffector function.

In a preferred embodiment the effector function is mediated by an F_(c)domain.

In a preferred embodiment the CD33 binding agent comprises one or moremutations in the F_(c) domain that modulate the function of the F_(c)domain.

In a preferred embodiment the modulation of the function of the F_(c)domain is an increase of ADCC by at least 10%, preferably 50% or 100%.

Particularly preferred CD33 binding agents according to the presentinvention are listed in table 1:

TABLE 1 SeqID SeqID Clone SeqID SeqID SeqID SeqID SeqID SeqID SeqIDSeqID heavy light No ID# CDR1 CDR2 CDR3 CDR4 CDR5 CDR6 V_(H) V_(L) chainchain 1 280- 29 15 1 71 57 43 85 99 113 127 03-08 2 280- 30 16 2 72 5844 86 100 114 128 21-09 3 280- 31 17 3 73 59 45 87 101 115 129 29-12 4280- 32 18 4 74 60 46 88 102 116 130 31-01 5 280- 33 19 5 75 61 47 89103 117 131 31- 01 (m ut) 6 280- 34 20 6 76 62 48 90 104 118 132 34-02 7280- 35 21 7 77 63 49 91 105 119 133 50-01 8 280- 36 22 8 78 64 50 92106 120 134 50- 01 (m ut) 9 280- 37 23 9 79 65 51 93 107 121 135 61-0710 283- 38 24 10 80 66 52 94 108 122 136 03-03 11 283- 39 25 11 81 67 5395 109 123 137 05-01 12 283- 40 26 12 82 68 54 96 110 124 138 07-03 13283- 41 27 13 83 69 55 97 111 125 139 11-03 14 283- 42 28 14 84 70 56 98112 126 140 14-01

Treatment of Cancer

Several publications have described CD33 as a cell surface marker onprimary AML and CML cells which is expressed on malignant cells of 70%to 100% of patients (Scheinberg et al 1989, Hauswirt et al 2007, Plesaet al 2007, Webber et al 2008). CD33 is expressed on malignant myeloidblast cells, which represent the majority of malignant cells inperipheral blood and bone marrow of leukemia patients, and on leukemicstem cells, a relatively small number of less differentiated cells inthe bone marrow which are characterized by their capacity forself-renewal and the maintenance of the leukemic clonal hierarchy. Theclinical feasibility of targeting CD33 using antibodies has beendemonstrated by Mylotarg® (Gemtuzumab ozogamizin), anantibody-calicheamycin conjugate which was approved for the treatment ofrelapsed AML patients not eligible for other treatment options. Analternative approach directed towards CD33 is the development oflintuzumab (SGN-33, HuM195), a humanized IgG1 monoclonal antibody whichhas shown early signs of efficacy in clinical phase I trials (Raza etal., 2009). Taken together, there is a wealth of data, both preclinicaland clinical, that underscore the relevance and feasibility of CD33targeting for the treatment of AML and other CD33-positive malignancies.

Acute myeloid leukemia (AML) is a malignancy of the myeloid lineage ofwhite blood cells. This hematological neoplasia is a blood and bonemarrow disease that, if left untreated, is typically fatal within weeksto months. There is an estimated prevalence of 30000 cases of AML in theUS and 47000 in the European Union (10 year prevalence data confirmed byMattson-Jack, 2010). AML is the most prevalent form of adult acuteleukemia (about 90%), comprising about 33% of new leukemia cases. Themedian age of patients diagnosed with AML is 67 years. AML accounts forabout 1.2% of cancer deaths in the United States.

AML causes non-specific symptoms such as weight loss, fatigue, fever,and night sweats. AML is diagnosed by blood tests, bone marrowexamination, and laboratory tests to determine the AML subtype and todetermine treatment decisions.

Therapy for AML is highly depending on the age and performance status ofthe patient. Patients who can tolerate intensive induction (andsubsequent consolidation and maintenance) chemotherapy will be treatedintensely with a combination of cytotoxic drugs. These patients have alikelihood of achieving a complete response of approximately 75%. Inthis patient population the therapeutic goal is a cure. Still, relapseof AML occurs in about half of the patients within one year afterachievement of a complete response. Long-term cure rates are in the 30%range.

However, higher age at diagnosis or existence of co-morbidities does notallow administration of intensive induction therapy leading to apalliative treatment goal. Therefore, remission rates declinesignificantly in older patients with AML. The median survival of olderpatients with AML is less than 6 months.

In one aspect, the CD33 binding agents are useful for treating cancer,such as by delaying progression of a cancer and/or reducingcancer-associated cachexia, or preventing or delaying recurrence of ahematological malignancy (e.g., leukemia), in a mammal, preferably ahuman patient. The CD33 binding agent can be administered alone orco-administered with another therapeutic agent. In some embodiments, theCD33 binding agent is co-administered with a standard of carechemotherapeutic. The CD33 binding agent can be administered in anunconjugated form (i.e., not conjugated to a cytotoxin) or as aconjugate.

In this subsection, a “patient” is a human or other mammal who isundergoing treatment for, or has been diagnosed as having, cancer.

In some embodiments, the CD33 binding agents are useful for delayingprogression of a cancer and/or reducing cancer-associated cachexia in apatient by administering to the patient in need thereof an effectivedosage of the CD33 binding agent. Without being bound to a particularmechanism, the CD33 binding agent binds to effector or accessory cellsof the myeloid or monocytic lineages (e.g., monocytes, macrophages,dendritic cells, and neutrophils), thereby inhibiting or reducing theproduction of various cytokines, chemokines and growth factors from theeffector or accessory cells and/or the tumor cells. These cytokines,chemokines and growth factors, which can promote the growth andproliferation of tumor cells and/or contribute to cancer cachexia,include, but are not limited to, interleukin-1β (IL-1β), tumor necrosisfactor-α (TNF-α), interleukin-6 (IL-6), interleukin-8 (IL-8),interferon-γ (IFN-γ), vascular endothelial growth factor (VEGF),leukemia inhibitory factor (L1F), monocyte chemoattractant protein-1(MCP-1), RANTES, interleukin-10 (IL-10), interleukin-12 (IL-12), matrixmetalloproteinase 2 (MMP2), IP-10 and/or macrophage inflammatory protein1a (MIP1α). The CD33 binding agents also can reduce the migration ofmacrophages to the site of the tumor cells.

In some embodiments, administration of an effective dosage of a CD33binding agent to a patient reduces the levels of at least one cytokine,chemokine or growth factor, which cytokine, chemokine or growth factorcan promote the growth and proliferation of tumor cells, promote themigration of non-malignant effector cells, such as tumor associatedmacrophages (TAMS), to the vicinity of the tumor site and/or contributeto cancer cachexia. In specific embodiments, the cytokine, chemokine orgrowth factor is for example interleukin-1β (IL-1β), tumor necrosisfactor-α (TNF-α), interleukin-6 (IL-6), interleukin-8 (IL-8),interferon-γ (IFN-γ), vascular endothelial growth factor (VEGF),leukemia inhibitory factor (LIF), monocyte chemoattractant protein-1(MCP-1), RANTES, interleukin-10 (IL-10), interleukin-12 (IL-12), matrixmetalloproteinase 2 (MMP2), IP-10 and/or macrophage inflammatory protein1a (MIP1 a).

In another embodiment, a method is provided for delaying progression ofa cancer by administering to a patient an effective regimen of a CD33binding agent that can specifically bind to CD33. As a result ofadministration of the CD33 binding agent, the progression of the canceris delayed, such as by reducing the growth or proliferation of the tumorcells, decreasing metastasis, reducing the level of at least onecytokine, chemokine or growth factor, reducing non-malignant effectorcells in the vicinity of the tumor cells, or the like.

In another embodiment, a method is provided for reducing the tumorburden in a patient by administering to the patient an effective regimenof a CD33 binding agent that can specifically bind to CD33. As a resultof administration of the CD33 binding agent, the tumor burden in thepatient is arrested or reduced, such as by reducing the size or mass ofthe tumor, reducing the level of at least one cytokine, chemokine orgrowth factor, reducing non-malignant effector cells in the vicinity ofthe tumor cells, inhibiting the migration of macrophages in the vicinityof the tumor cells, reducing the number of non-malignant effector cells(e.g., TAMS or macrophages) in the tumor, or the like.

In another embodiment, a method is provided for reducing the tumorburden or delaying progression of a cancer in a patient by administeringto the patient an effective regimen of a CD33 binding agent that canspecifically bind to CD33. As a result of administration of the CD33binding agent, the tumor burden in the patient is arrested or reduced,such as by recruiting immune effector cells like NK cells or macrophagesor monocytes, which can destroy tumor cells by immune mediatedmechanisms.

Antibody dependent cellular cytotoxicity (ADCC) is an immune effectorcell mediated mechanism which may contribute to anti-tumor activity ofmonoclonal antibodies (Weiner G J. Monoclonal antibody mechanisms ofaction in cancer. Immunol Res. 2007; 39(1-3):271-8). The relevance ofADCC for anti-tumor efficacy has been demonstrated in preclinicalmodels, e.g. in mouse tumor models (e.g. Clynes R A, Towers T L, PrestaL G, Ravetch J V. Inhibitory Fc receptors modulate in vivo cytoxicityagainst tumor targets. Nat Med. 2000 April; 6(4):443-6). Data fromclinical trials support the relevance of ADCC for clinical efficacy oftherapeutic antibodies (e.g. Weng W K, Levy R Two immunoglobulin Gfragment C receptor polymorphisms independently predict response torituximab in patients with follicular lymphoma. J Clin Oncol. 2003 Nov.1; 21(21):3940-7. Epub 2003 Sep. 15). Interactions of monoclonalantibodies with Fc receptors on immune cells contribute to ADCC. The Fcof antibodies can be modified in order to display enhanced affinity toFc receptors (e.g. Presta LG Engineering of therapeutic antibodies tominimize immunogenicity and optimize function. Adv Drug Deliv Rev. 2006Aug. 7; 58(5-6):640-56. Epub 2006 May 23). Such enhanced affinity to Fcreceptors results in increased ADCC activity which may lead to increasedanti tumor efficacy in patients.

In the various embodiments described in this section, the CD33 bindingagent can be used to treat a CD33 positive cancer (i.e., a cancercomprised of cancer cells that over express CD33 on their cell surface,or that express CD33 at levels considered acceptable for therapy withCD33 antibodies). The CD33 binding agent also can be used to treat acancer that does not overexpress CD33 on the non-malignant effectorcells relative to normal tissue of the same type. The cancer can be, forexample, a non-hematological malignancy or a hematological malignancy.In specific examples, the hematological malignancy can be CD33-positiveand can be, for example, acute lymphoid leukemia, acute myeloidleukemia, chronic myelomonocytic leukemia, an erythrocytic leukemia,acute megakaryoblastic leukemia, histiocytic lymphoma, a myeloidsarcoma, a mast cell proliferative disorder or myelodysplastic syndrome(MDS). In some embodiments, the hematological malignancy is aCD33-positive malignancy, such as acute myeloid leukemia ormyelodysplastic syndrome (MDS).

In the various embodiments described in this section, the CD33 bindingagent can be an unconjugated anti-CD33 antibody. For example, theantibody can be a fully human, humanized or chimeric antibody, such as achimeric or humanized M 195 antibody. The antibody also can be anotherantibody, such as an antibody that competes with M 195 antibody forspecific binding to CD33. The antibody also can bind to the same epitopeas M 195 antibody or to a different epitope.

In other embodiments, the CD33 binding agent can be bound (i.e.,conjugated) to a cytotoxin. The cytotoxin can be, for example, a peptidetoxin, such as saporin, ricin, chlorotoxin, Pseudomonas exotoxin,Pseudomonas endotoxin or diphtheria toxin. The cytotoxin also can be achemical (i.e., non-peptide-based) toxin, such as a calicheamicin,doxorubicin, a camptothecin, daunorubicin, or other DNA binding agents.The cytotoxin also can be an auristatin, a maytansinoid, a dolastatin,or other microtubule blocking agents.

A CD33 binding agent that is an anti-CD33 antibody can be administeredto the patient intravenously or subcutaneously at a dose of 0.1 mg/kg orless to about 25 mg/kg, preferably 1.0 mg/kg to about 10 mg/kg. A CD33binding agent that is an anti-CD33 antibody fragment or other CD33binding protein can be administered in a dosage equivalent to a dose of0.1 mg/kg to about 25 mg/kg. 1.0 mg/kg to about 10 mg/kg of intactantibody. The CD33 binding agent can be administered intravenously orsubcutaneously to the patient on a schedule that is, for example, daily,weekly, biweekly, tri-weekly (i.e., every three weeks) or monthly, or acombination thereof, to the patient. The CD33 binding agent can beadministered for a period of at least one month, at least two months, atleast three months, at least four months, at least five month, at leastsix months, or more, as needed. In some embodiments, a treatment phase(supra) of the CD33 binding agent is followed by a maintenance phase, inwhich doses of the CD33 binding agent are administered less frequentlythan during the treatment phase. For example, maintenance doses can beadministered weekly, biweekly, tri-weekly or monthly, for a period of1-6 months. The dosages in the maintenance phase can be the same as thedosages in the treatment phase.

Treatment of a Hematological Malignancy in Remission

In another aspect, methods are provided of preventing or delayingrecurrence of a hematological malignancy (e.g., leukemia) in a patientby administering to the patient in remission from the hematologicalmalignancy an effective dosage of a CD33 binding agent, resulting inpreventing or delaying recurrence of the underlying hematologicalmalignancy. The CD33 binding agent specifically binds to CD33 on thesurface of the hematological malignancy (i.e., leukemic cell) and/or tonon-malignant effector cells.

In this disclosure, a “patient” is typically a human who is undergoingtreatment for, or has been diagnosed as having, hematologicalmalignancy. In some embodiments, the hematological malignancy is aCD33-positive hematological malignancy. Hematological malignanciesinclude, but are not limited to, leukemias (e.g., acute lymphoblasticleukemia (ALL), acute myelogenous leukemia (AML), chronic myelogenousleukemia (CML), chronic myelomonocytic leukemia, hairy cell leukemia),Related blood disorders include, but are not limited to, myelodysplasticsyndrome (MDS), myelofibrosis, myeloproliferative disease (e.g.,polycythemia vera (PV. PCV or PRV), essential thrombocytosis (ET)), andamyloid due to light-chain disease.

The term “CD33-positive hematological malignancy” refers to ahematological malignancy characterized by the expression of CD33 on thesurface of the malignant cells. CD33-positive hematological malignanciesinclude, but are not limited to, acute myeloid leukemia (AML), chronicmyeloid leukemia (CML), chronic myelomonocytic leukemia, thrombocyteleukemia, a myelodysplastic syndrome, a myeloproliferative disorder,refractory anemia, a preleukemia syndrome, a lymphoid leukemia, or anundifferentiated leukemia.

In some embodiments, the methods include administering to a patient inremission from a CD33 positive hematological malignancy an effectiveregimen of a CD33 binding agent, whereby the recurrence of thehematological malignancy is prevented or delayed. In some embodiments,the patient lacks detectable cells of the hematological malignancy. Asused herein, a ‘lack of detectable cells” is determined by standarddiagnostic or prognostic methods. A patient in remission from AMLtypically exhibits resolution of abnormal clinical features, return tonormal blood counts and normal hematopoiesis in the bone marrow with <5%blast cells, a neutrophil count of >1.000-1,500, a platelet countof >100,000, and disappearance of the leukemic clone. See, e.g., TheMerck Manual, Sec. 11, Ch. 138 (17^(th) ed. 1997): Estey, 2001, Cancer92(5): 1059-1073.

The CD33 binding agent can be, for example, an antibody thatspecifically binds to CD33 and the hematological malignancy can be acutemyeloid leukemia (AML), chronic myeloid leukemia (CML), chronicmyelomonocytic leukemia, thymoid leukemia, a myelodysplastic syndrome, amyeloproliferative disorder, refractory anemia, a preleukemia syndrome,a lymphoid leukemia, or an undifferentiated leukemia.

In some embodiments, the patient in remission from the hematologicalmalignancy has not undergone a bone marrow transplant. In otherembodiments, the patient in remission from the hematological malignancyhas undergone a bone marrow transplant. The bone marrow transplant canbe either an autologous or an allogeneic bone marrow transplant.

The following cancer types are especially suitable to be treated by theantibodies according to the present invention:

Blood-borne cancers, including, but not limited to: acute lymphoblasticleukemia “ALL”, acute lymphoblastic B-cell leukemia, acute lymphoblasticT-cell leukemia, acute myeloblastic leukemia “AML”, acute promyelocyticleukemia “APL”, acute monoblastic leukemia, acute erythroleukemicleukemia, acute megakaryoblaslic leukemia, acute myelomonocyticleukemia, acute nonlymphocytic leukemia, acute undifferentiatedleukemia, chronic myelocytic leukemia “CML”, chronic lymphocyticleukemia “CLL”, hairy cell leukemia, multiple myeloma.

Acute and chronic leukemias, which are suitable to be treated by CD33binding agents include: lymphoblastic, myelogenous, lymphocytic,myelocytic leukemias and thrombocytic leukemia. Further myelodysplasiasyndrome, myeloproliferative disorder, refractory anemia, preleukemiasyndrome, lymphoid leukemia, or undifferentiated leukemia can be treatedby CD33 binding agents.

Combinations with Other Active Substances

Depending on the disorder to be treated, the CD33 binding agents of theinvention may be used on its own or in combination with one or moreadditional therapeutic agents, in particular selected from DNA damaging,DNA demethylating or tubulin binding agents or therapeutically activecompounds that inhibit angiogenesis, signal transduction pathways ormitotic checkpoints in cancer cells or have immunomodulatory function(IMIDs).

The additional therapeutic agent may be administered simultaneouslywith, optionally as a component of the same pharmaceutical preparation,or before or after administration of the CD33 binding agent.

In certain embodiments, the additional therapeutic agent may be, withoutlimitation, one or more inhibitors selected from the group of inhibitorsof EGFR family, VEGFR family, VEGF, IGF-1R, Insulin receptors, AuroraA,AuroraB, PLK and PI3 kinase, FGFR, PDGFR, Raf, KSP or PDK1.

Further examples of additional therapeutic agents are inhibitors ofCDKs, Akt, Src, Bcr-Abl, cKit, cMet/HGF, Her2, Her3, c-Myc, Flt3, HSP90,hedgehog antagonists, inhibitors of JAK/STAT, Mek, mTor, NFkappaB, theproteasome, Rho, an inhibitor of Wnt signaling or Notch signaling or anubiquitination pathway inhibitor.

Further examples of additional therapeutic agents are inhibitors of DNApolymerase, topoisomerase II, multityrosine kinase inhibitors, CXCR4antagonists, IL3RA inhibitors, RAR antagonists, KIR inhibitors,immunotherapeutic vaccines, TUB inhibitors, Hsp70 inducers, IAP familyinhibitors, DNA methyltransferase inhibitors, TNF inhibitors, ErbB1receptor tyrosine kinase inhibitors, multikinase inhibitors, JAK2inhibitors, RR inhibitors, apoptosis inducers, HGPRTase inhibitors,histamine H2 receptor antagonists and CD25 receptor agnosists.

Examples for Aurora inhibitors are, without limitation, PHA-739358,AZD-1152, AT-9283, CYC-116, R-763, VX-667, MLN-8045, PF-3814735,SNS-314, VX-689, GSK-1070916, TTP-607, PHA-680626, MLN-8237, BI847325and ENMD-2076.

Examples for PLK inhibitor are GSK-461364, BI2536 and BI6727.

Examples for raf inhibitors are BAY-73-4506 (also a VEGFR inhibitor),PLX-4032, RAF-265 (also a VEGFR inhibitor), sorafenib (also a VEGFRinhibitor), XL-281, Nevavar (also an inhibitor of the VEGFR) andPLX4032.

Examples for KSP inhibitors are ispinesib, ARRY-520, AZD-4877,CK-1122697, GSK-246053A, GSK-923295, MK-0731, SB-743921, LY-2523355, andEMD-534085.

Examples for a src and/or bcr-abl inhibitors are dasatinib, AZD-0530,bosutinib, XL-228 (also an IGF-1R inhibitor), nilotinib (also a PDGFRand cKit inhibitor), imatinib (also a cKit inhibitor), NS-187, KX2-391,AP-24534 (also an inhibitor of EGFR, FGFR, Tie2, Flt3), KM-80 and LS-104(also an inhibitor of Flt3, Jak2).

An example for a PDK1 inhibitor is AR-12.

An example for a Rho inhibitor is BA-210.

Examples for PI3 kinase inhibitors are PX-866, PX-867, BEZ-235 (also anmTor inhibitor), XL-147, and XL-765 (also an mTor inhibitor), BGT-226,CDC-0941.

Examples for inhibitors of cMet or HGF are XL-184 (also an inhibitor ofVEGFR, cKit, Flt3), PF-2341066, MK-2461, XL-880 (also an inhibitor ofVEGFR), MGCD-265 (also an inhibitor of VEGFR, Ron, Tie2), SU-11274,PHA-665752, AMG-102, AV-299, ARQ-197, MetMAb, CGEN-241, BMS-777607,JNJ-38877605, PF-4217903, SGX-126, CEP-17940, AMG-458, INCB-028060, andE-7050.

An example for a c-Myc inhibitor is CX-3543.

Examples for Flt3 inhibitors are AC-220 (also an inhibitor of cKit andPDGFR), KW-2449, LS-104 (also an inhibitor of bcr-abl and Jak2),MC-2002, SB-1317, lestaurtinib (also an inhibitor of VEGFR, PDGFR, PKC),TG-101348 (also an inhibitor of JAK2), XL-999 (also an inhibitor ofcKit, FGFR, PDGFR and VEGFR), sunitinib (also an inhibitor of PDGFR,VEGFR and cKit), and tandutinib (also an inhibitor of PDGFR, and cKit).

Examples for HSP90 inhibitors are, tanespimycin, alvespimycin, IPI-504,STA-9090, MEDI-561, AUY-922, CNF-2024, and SNX-5422.

Examples for JAK/STAT inhibitors are CYT-997 (also interacting withtubulin), TG-101348 (also an inhibitor of Flt3), and XL-019.

Examples for Mek inhibitors are ARRY-142886, AS-703026, PD-325901,AZD-8330, ARRY-704, RDEA-119, and XL-518.

Examples for mTor inhibitors are temsirolimus, deforolimus (which alsoacts as a VEGF inhibitor), everolimus (a VEGF inhibitor in addition).XL-765 (also a PI3 kinase inhibitor), and BEZ-235 (also a PI3 kinaseinhibitor).

Examples for Akt inhibitors are perifosine, GSK-690693, RX-0201, andtriciribine.

Examples for cKit inhibitors are masitinib, OSI-930 (also acts as aVEGFR inhibitor), AC-220 (also an inhibitor of Flt3 and PDGFR),tandutinib (also an inhibitor of Flt3 and PDGFR), axitinib (also aninhibitor of VEGFR and PDGFR), sunitinib (also an inhibitor of Flt3,PDGFR, VEGFR), and XL-820 (also acts as a VEGFR- and PDGFR inhibitor),imatinib (also a bcr-abl inhibitor), nilotinib (also an inhibitor ofbcr-abl and PDGFR).

Examples for hedgehog antagonists are IPI-609, CUR-61414, GDC-0449,IPI-926, and XL-139.

Examples for CDK inhibitors are seliciclib, AT-7519, P-276, ZK-CDK (alsoinhibiting VEGFR2 and PDGFR), PD-332991, R-547, SNS-032, PHA-690509,PHA-848125, and SCH-727965.

Examples for proteasome inhibitors are bortezomib, carfilzomib, andNPI-0052 (also an inhibitor of NFkappaB).

Examples for proteasome inhibitors/NFkappaB pathway inhibitors arebortezomib, carfilzomib, NPI-0052, CEP-18770, MLN-2238, PR-047, PR-957,AVE-8680, and SPC-839.

An example for an inhibitor of the ubiquitination pathway is HBX-41108.

Examples for demethylating agends are 5-azacitidine and decitabine.

Examples for anti-angiogenic agents are inhibitors of the FGFR, PDGFRand VEGF(R), and thalidomides, such agents being selected from, withoutlimitation, bevacizumab, motesanib, CDP-791, SU-14813, telatinib,KRN-951, ZK-CDK (also an inhibitor of CDK), ABT-869, BMS-690514,RAF-265, IMC-KDR, IMC-18F1, IMiDs, thalidomide, CC-4047, lenalidomide,ENMD-0995, IMC-D11, Ki-23057, brivanib, cediranib, 1B3, CP-868596,IMC-3G3, R-1530 (also an inhibitor of Flt3), sunitinib (also aninhibitor of cKit and Flt3), axitinib (also an inhibitor of cKit),lestaurtinib (also an inhibitor of Flt3 and PKC), vatalanib, tandutinib(also an inhibitor of Flt3 and cKit), pazopanib, PF-337210, aflibercept,E-7080, CHIR-258, sorafenib tosylate (also an inhibitor of Rae,vandetanib, CP-547632, OSI-930, AEE-788 (also an inhibitor of EGFR andHer2), BAY-57-9352 (also an inhibitor of Raf), BAY-73-4506 (also aninhibitor of Raf), XL-880 (also an inhibitor of cMet), XL-647 (also aninhibitor of EGFR and EphB4), XL-820 (also an inhibitor of cKit),nilotinib (also an inhibitor of cKit and brc-abl), CYT-116, PTC-299,BMS-584622, CEP-11981, dovitinib, CY-2401401, ENMD-2976 and BIBF1120.

The additional therapeutic agent may also be selected from EGFRinhibitors, it may be a small molecule EGFR inhibitor or an anti-EGFRantibody. Examples for anti-EGFR antibodies, without limitation, arecetuximab, panitumumab, nimotuzumab, zalutumumab; examples for smallmolecule EGFR inhibitors are gefitinib, erlotinib, vandetanib (also aninhibitor of the VEGFR) and afatinib (also an inhibitor of Her2).Another example for an EGFR modulator is the EGF fusion toxin.

Further EGFR and/or Her2 inhibitors useful for combination with an CD33binding agent of the invention are lapatinib, trastuzumab, pertuzumab,XL-647, neratinib, BMS-599626 ARRY-334543, AV-412, mAB-806, BMS-690514,JNJ-26483327, AEE-788 (also an inhibitor of VEGFR), AZD-8931, ARRY-380ARRY-333786, IMC-11F8, Zemab, TAK-285, AZD-4769, and afatinib (dualinhibitor of Her2 and EGFR).

DNA polymerase inhibitors useful in the combination with an CD33 bindingagent of the invention are Ara-C/cytarabine, Clolar/clofarabine.

A DNA methyltransferase inhibitor useful in the combination with an CD33binding agent of the invention is Vidaza/azacitidine.

An apoptosis inducer useful in the combination with an CD33 bindingagent of the invention is Trisenox/arsenice trioxide.

Topoisomerase II inhibitors useful in the combination with an CD33binding agent of the invention are idarubicin, daunorubicin andmitoxantrone.

A RAR antagonist useful in the combination with an CD33 binding agent ofthe invention is Vesanoid/tretinoin.

A HGPRTase inhibitor useful in the combination with an CD33 bindingagent of the invention is Mercapto/mercaptopurine.

A histamine H2 receptor antagonist useful in the combination with anCD33 binding agent of the invention is Ceplene/histaminedihydrochloride.

A CD25 receptor agonist useful in the combination with an CD33 bindingagent of the invention is IL-2.

The additional drug may also be selected from agents that target theIGF-1R and insulin receptor pathways. Such agents include antibodiesthat bind to IGF-1R (e.g. CP-751871, AMG-479, IMC-Al2, MK-0646,AVE-1642, R-1507, BBB-022, SCH-717454, rhu Mab IGFR and novel chemicalentities that target the kinase domain of the IGF1-R (e.g. OSI-906 orBMS-554417, XL-228, BMS-754807).

Other agents that may be advantageously combined in a therapy with theCD33 binding agent of the invention are molecules targeting CD20,including CD20 specific antibodies like rituximab, LY-2469298,ocrelizumab, MEDI-552, IMMU-106, GA-101 (=R7159), XmAb-0367, ofatumumab, radiolabbeled CD20 antibodies, like tositumumab andibritumomab tiuxetan or other CD20 directed proteins, like the SMIPTru015, PRO-131921, FBT-A05, veltuzumab, R-7159.

CD33 binding agents may be combined with inhibitors of other surfaceantigens expressed on leukocytes, in particular antibodies orantibody-like molecules, e.g. anti-CD2 (siplizumab), anti-CD4(zanolimumab), anti-CD19 (MT-103, MDX-1342, SAR-3419, XmAb-5574),anti-CD22 (epratuzumab), anti-CD23 (lumiliximab), anti-CD30(iralumumab), anti-CD32B (MGA-321), anti-CD38 (HuMax-CD38), anti-CD40(SGN40), anti-CD52 (alemtuzumab), anti-CD80 (galiximab).

Other agents to be combined with CD33 binding agents are immunotoxinslike BL-22 (an anti-CD22 immunotoxin), inotuzumab ozogamicin (ananti-CD23 antibody-calicheamicin conjugate), RFT5.dgA (anti-CD25 Ricintoxin A-chain), SGN-35 (an anti-CD30-auristatin E conjugate), andgemtuzumab ozogamicin (an anti-CD33 calicheamicin conjugate), MDX-1411(anti-CD70 conjugate), or radiolabelled antibodies like ⁹⁰Y-epratuzumab(anti-CD22 radioimmunoconjugate).

In addition, CD33 binding agents may be combined with immunomodulators,agents, e.g. antibodies, that induce apoptosis or modify signaltransduction pathways like the TRAIL receptor modulators mapatumumab (aTRAIL-1 receptor agonist), lexatumumab (a TRAIL-2 receptor agonist),tigatuzumab, Apomab, AMG-951 and AMG-655; an anti-HLA-DR antibody (like1D09C3), an anti-CD74, an osteoclast differentiation factor ligandinhibitor (like denosumab), a BAFF antagonist (like AMG-623a) or anagonist of a Toll-like receptor (e.g. TLR-4 or TLR-9).

Other drugs that may be used in combination with the CD33 binding agentsof the present invention are selected from, but not limited to hormones,hormonal analogues and antihormonals (e.g. tamoxifen, toremifene,raloxifene, fulvestrant, megestrol acetate, flutamide, nilutamide,bicalutamide, cyproterone acetate, finasteride, buserelin acetate,fludrocortinsone, fluoxymesterone, medroxyprogesterone,hydroxyprogesterone caproate, diethylstilbestrol, testosteronepropionate, fluoxymesterone/equivalents, octreotide, arzoxifene,pasireotide, vapreotide, adrenocorticosteroids/antagonists, prednisone,dexamethasone, ainoglutethimide), aromatase inhibitors (e.g.anastrozole, letrozole, liarozole, exemestane, atamestane, formestane),LHRH agonists and antagonists (e.g. goserelin acetate, leuprolide,abarelix, cetrorelix, deslorelin, histrelin, triptorelin),antimetabolites (e.g. antifolates like methotrexate, trimetrexate,pemetrexed, pyrimidine analogues like 5-fluorouracil,fluorodeoxyuridine, capecitabine, decitabine, nelarabine, 5-azacytidine,and gemcitabine, purine and adenosine analogues such as mercaptopurine,thioguanine, azathioprine, cladribine and pentostatin, cytarabine,fludarabine, clofarabine); antitumor antibiotics (e.g. anthracyclineslike doxorubicin, daunorubicin, epirubicin and idarubicin, mitomycin-C,bleomycin dactinomycin, plicamycin, splicamycin, actimomycin D,mitoxantrone, mitoxantroneidarubicin, pixantrone, streptozocin,aphidicolin); platinum derivatives (e.g. cisplatin, oxaliplatin,carboplatin, lobaplatin, satraplatin); alkylating agents (e.g.estramustine, semustine, mechlorethamine, melphalan, chlorambucil,busulphan, dacarbazine, cyclophosphamide, ifosfamide, hydroxyurea,temozolomide, nitrosoureas such as carmustine and lomustine, thiotepa);antimitotic agents (e.g. vinca alkaloids like vinblastine, vindesine,vinorelbine, vinflunine and vincristine; and taxanes like paclitaxel,docetaxel and their formulations, larotaxel; simotaxel, and epothiloneslike ixabepilone, patupilone, ZK-EPO); topoisomerase inhibitors (e.g.epipodophyllotoxins like etoposide and etopophos, teniposide, amsacrine,topotecan, irinotecan, banoxantrone, camptothecin) and miscellaneouschemotherapeutics such as retinoic acid derivatives, amifostine,anagrelide, interferon alpha, interferon beta, interferon gamma,interleukin-2, procarbazine, N-methylhydrazine, mitotane, and porfimer,bexarotene, celecoxib, ethylenemine/methyl-melamine,thriethyienemelamine, triethylene thiophosphoramide, hexamethylmelamine,and enzymes L-asparaginase, L-arginase and metronidazole, misonidazole,desmethylmisonidazole, pimonidazole, etanidazole, nimorazole, RSU 1069,EO9, RB 6145, SR4233, nicotinamide, 5-bromodeozyuridine,5-iododeoxyuridine, bromodeoxycytidine, erythrohydroxynonyl-adenine,anthracenedione, GRN-163L (a competitive telomerase templateantagonist), SDX-101 (a PPAR agonist), talabostat (a DPP inhibitor),forodesine (a PNP inhibitor), atacicept (a soluble receptor targetingTNF family members BLyS and APRIL), TNF-alpha neutralizing agents(Enbrel, Humira, Remicade), XL-844 (a CHK1/2 inhibitor), VNP-40101M (aDNA alkylating agent), SPC-2996 (an antisense bc12 inhibitor), obatoclax(a bcl2 inhibitor), enzastaurin (a PKC beta modulator), vorinistat (anHDAC inhibitor), romidepsin (an HDAC inhibitor), AT-101 (a Bcl-2/Bcl-xLinhibitor), plitidepsin (a multi-actioned depsipeptide), SL-11047 (apolyamine metabolism modulators).

The CD33 binding agents of the invention may also be used in combinationwith other therapies including surgery, stem cell transplantation,radiotherapy, endocrine therapy, biologic response modifiers,hyperthermia and cryotherapy and agents to attenuate any adverse effect(e.g. antiemetics), G-CSF, GM-CSF, photosensitizers such ashematoporphyrin derivatives, Photofrin, benzoporphyrin derivatives,Npe6, tin etioporphyrin, pheoboride-a bacteriochlorophyll-a,naphthalocyanines, phthalocyanines, zinc phthalocyanines.

Pharmaceutical Compositions and Methods of Administration

The CD33 binding agents can be in any form that allows for thecomposition to be administered to a patient. For example, thecomposition can be in the form of a solid or liquid. The preferred modeof application is parenteral, by infusion or injection (intraveneous,intramuscular, subcutaneous, intraperitoneal, intradermal), but othermodes of application such as by inhalation, transdermal, intranasal,buccal, oral and intra-tumor may also be applicable. Parenteraladministration includes subcutaneous injections, intravenous,intramuscular, intrastemal injection or infusion techniques. In oneaspect, the compositions are administered parenterally. In yet anotheraspect, the compounds are administered intravenously.

Pharmaceutical compositions can be formulated so as to allow a compoundto be bioavailable upon administration of the composition to a patient.Compositions can take the form of one or more dosage units, where, forexample, a container of a compound in aerosol form can hold a pluralityof dosage units.

Materials used in preparing the pharmaceutical compositions can benon-toxic in the amounts used. It will be evident to those of ordinaryskill in the art that the optimal dosage of the active ingredient(s) inthe pharmaceutical composition will depend on a variety of factors.Relevant factors include, without limitation, the type of patient (e.g.,human), the particular form of the compound, the manner ofadministration, and the composition employed.

The pharmaceutically acceptable carrier or vehicle can be particulate,so that the compositions are, for example, in powder form. Thecarrier(s) can be liquid, with the compositions being, for example, aninjectable liquid. The composition can be in the form of a liquid, e.g.,for parenteral injection. In a composition for administration byinjection, one or more of a surfactant, preservative, wetting agent,dispersing agent, suspending agent, buffer, stabilizer and isotonicagent can also be included.

The liquid compositions, whether they are solutions, suspensions orother like form, can also include one or more of the following: sterilediluents such as water for injection, saline solution, preferablyphysiological saline, Ringer's solution, isotonic sodium chloride, fixedoils such as synthetic mono- or digylcerides which can serve as thesolvent or suspending medium, polyethylene glycols, glycerin,cyclodextrin, propylene glycol or other solvents; stabilizers such asamino acids; surfactants such as polysorbates; antibacterial agents suchas benzyl alcohol or methyl paraben; antioxidants such as ascorbic acidor sodium bisulfite; chelating agents such as ethylenediaminetetraacelicacid; buffers such as acetates, citrates or phosphates; and agents forthe adjustment of tonicity such as sodium chloride or dextrose. Aparenteral composition can be enclosed in ampoule, a disposable syringeor a multiple-dose vial made of glass, plastic or other material.Physiological saline is an exemplary adjuvant. An injectable compositionis preferably sterile.

The CD33 binding agents may also be dried (freeze-dried, spray-dried,spray-freeze dried, dried by near or supercritical gases, vacuum dried,air-dried), precipitated or crystallized or entrapped in microcapsulesthat are prepared, for example, by coacervation techniques or byinterfacial polymerization using, for example, hydroxymethylcellulose orgelatin and poly-(methylmethacylate), respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules), in macroemulsions orprecipitated or immobilized onto carriers or surfaces, for example bypcmc technology (protein coated microcrystals). Such techniques aredisclosed in Remington: The Science and Practice of Pharmacy, 21^(st)edition, Hendrickson R. Ed.

The amount of the composition that is effective in the treatment of aparticular disorder or condition will depend on the nature of thedisorder or condition, and can be determined by standard clinicaltechniques. In addition, in vitro or in vivo assays can optionally beemployed to help identify optimal dosage ranges. The precise dose to beemployed in the compositions will also depend on the route ofadministration, and the seriousness of the disease or disorder, andshould be decided according to the judgment of the practitioner and eachpatient's circumstances.

The compositions comprise an effective amount of a drug(s) or agent(s)such that a suitable dosage will be obtained. Typically, this amount isat least about 0.01% of a drug or agent by weight of the composition.When intended for oral administration, this amount can be varied torange from about 0.1% to about 80% by weight of the composition. In oneaspect, oral compositions can comprise from about 4% to about 50% of thecompound by weight of the composition. In yet another aspect, presentcompositions are prepared so that a parenteral dosage unit contains fromabout 0.01% to about 2% by weight of the compound.

For intravenous administration, the composition can comprise from about1 to about 50 mg of a drug or agent per kg of the patient's body weight.In one aspect, the composition can include from about I, 1.5 or 2.5 toabout 50 mg of a drug or agent per kg of the patient's body weight. Inanother aspect, the amount administered will be in the range from about1, 1.5 or 2.5 to about 25 mg/kg of body weight of a drug or agent.

In some embodiments, the dosage administered to a patient is less than0.1 mg/kg to about 50 mg/kg of the patient's body weight. (Forconversion to mg/mm², a BSA of 1.8 m² and a body weight of 80 kg can beused.)

As discussed herein, a CD33 binding agent can be administeredintravenously or subcutaneously to the patient on a schedule that is,for example, daily, weekly, biweekly, tri-weekly or monthly to thepatient. For example, a CD33 binding agent can be administered weekly,for a period of 2 to 10 weeks, typically 3-6 weeks. In some embodiments,the dosage regimen of the CD33 binding agent maintains a blood serumconcentration of antibody at least 5 μg/ml or at least 10 μg/ml duringthe dosage cycle. The CD33 binding agent can be administered, forexample, from 1-8, or more cycles. In some embodiments, a CD33 bindingagent is administered chronically to a subject.

By way of example, the invention includes a method of treating a cancer,such as myeloid leukemia, by administering 0.1 mg/kg to 50 mg/kg, forinstance about 1.5-8 or 2.5-8 mg/kg, of an anti-CD33 antibody accordingto the invention weekly.

This treatment can be usually be continued for about 1-3 months,typically about two months. In an embodiment, the dosing schedule ismaintained until a reduction in blasts is noted. For example, dosing canbe continued up to about 6 months. This treatment can be followed by aless frequent dosing schedule, involving for instance biweekly doses (ortwice per month). This dosing schedule can be maintained 1, 2, 3, 4, 5,6 months or more to maintain a reduction in blasts and/or a remission.

In some embodiments, a prophylactic agent can be administered with aCD33 binding agent to minimize infusion reactions. Suitable prophylacticagents include, for example, methyl prednisolone, diphenyldramine,acetaminophen or other suitable agent. The prophylactic agent can beadministered prior to or at about the same time as the CD33 bindingagent.

The drug(s) or agent(s) or compositions can be administered by anyconvenient route, for example, by infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, rectal and intestinal mucosa, etc.). Administration can besystemic or local. Various delivery systems are known, e.g.,encapsulation in liposomes, microparticles, microcapsules, capsules,etc., and can be used to administer a compound. In certain embodiments,more than one drug or agent or composition is administered to a patient.

It can be desirable to administer one or more drugs or agents orcompositions locally to the area in need of treatment, as appropriatefor the drug or agent. This can be achieved, for example, and not by wayof limitation, by local infusion during surgery; topical application,e.g., in conjunction with a wound dressing after surgery; by injection:by means of a catheter; by means of a suppository; or by means of animplant, the implant being of a porous, non-porous, or gelatinousmaterial, including membranes, such as sialastic membranes, or fibers.In one embodiment, administration can be by direct injection at the site(or former site) of a cancer, tumor or neoplastic or pre-neoplastictissue.

The drug(s) or agent(s) or compositions can be delivered in a controlledrelease system, such as a pump or various polymeric materials. In yetanother embodiment, a controlled-release system can be placed inproximity of the target of the drug(s) or agent(s) or compositions, thusrequiring only a fraction of the systemic dose (see, e.g., Goodson, inMedical Applications of Controlled Release, vol. 2, pp. 115-138 (1984)).Other controlled-release systems discussed in the review by Langer(1990, Science 249: 1527-1533) can be used.

The drugs or agents are formulated in accordance with routine proceduresas a pharmaceutical composition adapted for intravenous administrationto animals, particularly human beings, as appropriate for the drug oragent. Typically, the carriers or vehicles for intravenousadministration are sterile isotonic aqueous buffer solutions. Wherenecessary, the compositions can also include a solubilizing agent.Compositions for intravenous administration can optionally comprise alocal anesthetic such as lignocaine to ease pain at the site of theinjection. Generally, the ingredients are supplied either separately ormixed together in unit dosage form, for example, as a dry lyophilizedpowder or water-free concentrate in a hermetically sealed container suchas an ampoule or sachette indicating the quantity of active agent. Wheredrug or agent is to be administered by infusion, it can be dispensed,for example, with an infusion bottle containing sterile pharmaceuticalgrade water or saline. Where the drug or agent is administered byinjection, an ampoule of sterile water for injection or saline can beprovided so that the ingredients can be mixed prior to administration.

Compositions of therapeutic agents also can be administered according toaccepted dosage forms in the form of tablets, lozenges, aqueous or oilysuspensions, granules, powders, emulsions, capsules, syrups, or elixirs,for example. Orally administered compositions can contain one or moreoptional agents, for example, sweetening agents such as fructose,aspartame or saccharin; flavoring agents such as peppermint, oil ofwintergreen, or cherry; coloring agents; and preserving agents, toprovide a pharmaceutically palatable preparation. Moreover, where intablet or pill form, the compositions can be coated to delaydisintegration and absorption in the gastrointestinal tract therebyproviding a sustained action over an extended period of time.Selectively permeable membranes surrounding an osmotically activedriving compound are also suitable for orally administered drugs oragents. In these later platforms, fluid from the environment surroundingthe capsule is imbibed by the driving compound, which swells to displacethe agent or agent composition through an aperture. These deliveryplatforms can provide an essentially zero order delivery profile asopposed to the spiked profiles of immediate release formulations. Atime-delay material such as glycerol monostearate or glycerol stearatecan also be used.

The composition can include various materials that modify the physicalform of a solid or liquid dosage unit. For example, the composition caninclude materials that form a coating shell around the activeingredients. The materials that form the coating shell are typicallyinert, and can be selected from, for example, sugar, shellac, and otherenteric coating agents. Alternatively, the active ingredients can beencased in a gelatin capsule.

The compositions can be administered to a patient in need thereof at afrequency, or over a period of time, that is determined by the attendingphysician. The compositions can be administered over a period of 1 day,2 days, 3 days, 5 days, 7 days, 10 days, 14 days, 21 days, 28 days, onemonth, two months, or longer periods of time. It is understood that thecompositions can be administered for any period of time between 1 dayand two months or longer.

Production of Antibodies

Antibodies can be produced using any method useful for the synthesis ofantibodies, in particular, such as by recombinant expression or chemicalsynthesis.

Recombinant expression of antibodies, or fragments or derivativesthereof, typically involves construction of a nucleic acid that encodesthe antibody. If the nucleotide sequence of the antibody is known, anucleic acid encoding the antibody or a polypeptide thereof maybeassembled from chemically synthesized oligonucleotides (e.g., asdescribed in Kutmeier at al., 1994, BioTechniques 17:242), whichinvolves the synthesis of overlapping oligonucleotides containingportions of the sequence encoding the antibody, annealing and ligationof those oligonucleotides, and then amplification of the ligatedoligonucleotides, e.g., by PCR.

Alternatively, a nucleic acid molecule encoding an antibody or apolypeptide thereof can be generated from a suitable source. If a clonecontaining the nucleic acid encoding the particular antibody is notavailable, but the sequence of the antibody is known, a nucleic acidencoding the antibody can be obtained from a suitable source (e.g., anantibody cDNA library, or cDNA library generated from any tissue orcells expressing the immunoglobulin) by, e.g., PCR amplification usingsynthetic primers hybridizable to the 3′ and 5′ ends of the sequence orby cloning using an oligonucleotide probe specific for the particulargene sequence.

If an antibody that specifically recognizes a particular antigen is notcommercially available (or a source for a cDNA library for cloning anucleic acid encoding such an immunoglobulin is not available),antibodies specific for a particular antigen can be generated by anymethod known in the art, for example, by immunizing a patient, orsuitable animal model such as a rabbit or mouse, to generate polyclonalantibodies or, more preferably, by generating monoclonal antibodies,e.g., as described by Kohler and Milstein (1975, Nature 256:495-497) or,as described by Kozbor et al. (1983, Immunology Today 4:72) or Cole etal. (1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc., pp. 77-96). Alternatively, a clone encoding at least the Fabportion of the antibody can be obtained by screening Fab expressionlibraries (e.g., as described in Huse et al., 1989, Science 246,1275-1281) for clones of Fab fragments that bind the specific antigen orby screening antibody libraries (see, e.g., Clackson et al, 1991, Nature352:624; Hane et al, 1997, Proc. Natl. Acad. Sci. USA 94:4937).

Once a nucleic acid sequence encoding at least the variable domain ofthe antibody is obtained, it can be introduced into a vector containingthe nucleotide sequence encoding the constant regions of the antibody(see, e.g., International Publication No. WO 86/05807; WO 89/01036; andU.S. Pat. No. 5,122,464). Vectors containing the complete light or heavychain that allow for the expression of a complete antibody molecule areavailable. Then, the nucleic acid encoding the antibody can be used tointroduce the nucleotide substitution(s) or deletion(s) necessary tosubstitute (or delete) the one or more variable region cysteine residuesparticipating in an intrachain disulfide bond with an amino acid residuethat does not contain a sulfhydyl group. Such modifications can becarried out by any method known in the art for the introduction ofspecific mutations or deletions in a nucleotide sequence, for example,but not limited to, chemical mutagenesis and in vitro site directedmutagenesis (see, e.g., Hutchinson et al, 1978, J. Biol. Chem.253:6551).

In addition, techniques have been developed for the production of“chimeric antibodies” (see, e.g., Morrison et al., 1984, Proc. Natl.Acad. Sci. USA 81: 851-855; Neuberger et al., 1984, Nature 312:604-608;Takeda et al., 1985, Nature 314:452-454). A chimeric antibody is amolecule in which different portions are derived from different animalspecies, such as those having a variable region derived from a murinemonoclonal antibody and a human immunoglobulin constant region, e.g.,humanized antibodies.

Once a nucleic acid sequence encoding an antibody has been obtained, thevector for the production of the antibody can be produced by recombinantDNA technology using techniques known in the art. Methods that are knownto those skilled in the art can be used to construct expression vectorscontaining the antibody coding sequences and appropriate transcriptionaland translational control signals. These methods include, for example,in vitro recombinant DNA techniques, synthetic techniques, and in vivogenetic recombination. See, for example, the techniques described inSambrook et al (1990, Molecular Cloning, A Laboratory Manual. 2^(nd)Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; andSambrook et al., 2001; Molecular Cloning, A Laboratory Manual, 3^(rd)Ed., Cold Spring Harbor Publish., Cold Spring Harbor, N.Y.) and Ausubelet al. (eds., 1993-2006, Current Protocols in Molecular Biology, JohnWiley & Sons, NY).

An expression vector comprising the nucleotide sequence of an antibodyor the nucleotide sequence of an antibody can be transferred to a hostcell by conventional techniques (e.g., electroporation, liposomaltransaction, calcium phosphate precipitation or transduction), and theresulting cells are then cultured by conventional techniques to producethe antibody. In specific embodiments, the expression of the antibody isregulated by a constitutive, an inducible or a tissue-specific promoter.

The host cells used to express the recombinant antibody can be eitherbacterial cells such as Escherichia coli, or, preferably, eukaryoticcells, especially for the expression of recombinant immunoglobulinmolecules. In particular, mammalian cells such as Chinese hamster ovarycells (CHO), in conjunction with a vector containing the majorintermediate early gene promoter element from human cytomegalovirus isan effective expression system for immunoglobulins (see, e.g., Foeckinget al., 1986, Gene 45:101; Cockett et al., 1990, BioTechnology 8:2). TheCHO cell line can be, for example, DG44 or CHO-S. In another example, anantibody can be expressed using the CHEF system. (See, e.g., U.S. Pat.No. 5,888,809.)

A variety of other host-expression vector systems can be utilized toexpress antibodies. Such host-expression systems represent vehicles bywhich the coding sequences of the antibody can be produced andsubsequently purified, but also represent cells that can, whentransformed or transfected with the appropriate nucleotide codingsequences, express an antibody immunoglobulin molecule in situ. Theseinclude, but are not limited to, microorganisms such as bacteria (e.g.,E. coli and B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA or cosmid DNA expression vectors containing immunoglobulincoding sequences; yeast (e.g., Saccharomyces pichia) transformed withrecombinant yeast expression vectors containing antibody codingsequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing the immunoglobulincoding sequences; plant cell systems infected with recombinant virusexpression vectors (e.g., cauliflower mosaic virus (CaMV) and tobaccomosaic virus (TMV)) or transformed with recombinant plasmid expressionvectors (e.g., Ti plasmid) containing antibody coding sequences; ormammalian cell systems (e.g., COS, CHO, CHO-S, BH, 293, 293T or 3T3cells) harboring recombinant expression constructs containing promotersderived from the genome of mammalian cells (e.g., metallothioneinpromoter) or from mammalian viruses (e.g., the adenovirus late promoter;the vaccinia virus 7.5K promoter).

In bacterial systems, a number of expression vectors can beadvantageously selected depending upon the use intended for the antibodybeing expressed. For example, when a large quantity of such a protein isto be produced, vectors that direct the expression of high levels offusion protein products that are readily purified might be desirable.Such vectors include, but are not limited, to the E. coli expressionvector pUR278 (Ruther et al, 1983, EMBOJ. 2:1791-94), in which theantibody coding sequence may be ligated individually into the vector inframe with the lac Z coding region so that a fusion protein is produced;pIN vectors (Inouye and Inouye, 1985, Nucleic Acids Res. 13:3101-3109;Van Heeke and Schuster, 1989, J. Biol. Chem. 24:5503-5509); and thelike. pGEX vectors can also be used to express foreign polypeptides asfusion proteins with glutathione S-transferase (GST). In general, suchfusion proteins are soluble and can easily be purified from lysed cellsby adsorption and binding to a matrix glutathione-agarose beads followedby elution in the presence of free glutathione. The pGEX vectors aredesigned to include thrombin or factor Xa protease cleavage sites sothat the cloned target gene product can be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) or the analogous virus from Drosophila melanogaster can be usedas a vector to express foreign genes. The virus grows in Spodopierafrugipenta cells. The antibody coding sequence can be clonedindividually into non-essential regions (for example the polyhedringene) of the virus and placed under control of an AcNPV promoter (forexample the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems canbe utilized. In cases where an adenovirus is used as an expressionvector, the antibody coding sequence of interest can be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene can then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) results in a recombinant virus that is viable and capable ofexpressing the immunoglobulin molecule in infected hosts. (See, e.g.,Logan and Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:355-359). Specificinitiation signals can also be required for efficient translation ofinserted antibody coding sequences. These signals include the ATGinitiation codon and adjacent sequences. Furthermore, the initiationcodon is operably related to the reading frame of the desired codingsequence to ensure translation of the entire insert. These exogenoustranslational control signals and initiation codons can be of a varietyof origins, both natural and synthetic. The efficiency of expression canbe enhanced by the inclusion of appropriate transcription enhancerelements, transcription terminators, etc. (see, e.g., Bittner et al.,1987, Methods in Enzymol. 153:51-544).

In addition, a host cell strain can be chosen to modulate the expressionof the inserted sequences, or modify and process the gene product in thespecific fashion desired. Such modifications (e.g., glycosylation) andprocessing (e.g., cleavage) of protein products can be important for thefunction of the protein. Different host cells have characteristic andspecific mechanisms for the post-translational processing andmodification of proteins and gene products. Appropriate cell lines orhost systems can be chosen to ensure the correct modification andprocessing of the foreign protein expressed. To this end, eukaryotichost cells that possess the cellular machinery for proper processing ofthe primary transcript, glycosylation, and phosphorylation of the geneproduct can be used. Such mammalian host cells include, but are notlimited to, CHO (e.g., DG44 or CHO-S), VERY, BH, Hela, COS, MDCK, 293,293T, 3T3, W138, BT483, Hs578T, HTB2, BT20 and T47D, CRL7030 andHs578Bst.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines that stably express anantibody can be engineered. Rather than using expression vectors thatcontain viral origins of replication, host cells can be transformed withDNA controlled by appropriate expression control elements (e.g.,promoter, enhancer, sequences, transcription terminators,polyadenylation sites, etc.), and a selectable marker. Following theintroduction of the foreign DNA, engineered cells can be allowed to growfor 1-2 days in an enriched media, and then are switched to a selectivemedia. The selectable marker in the recombinant plasmid confersresistance to the selection and allows cells to stably integrate theplasmid into their chromosomes and grow to form foci that in turn can becloned and expanded into cell lines. This method can advantageously beused to engineer cell lines which express the antibody. Such engineeredcell lines can be particularly useful in screening and evaluation oftumor antigens that interact directly or indirectly with the antibody.

A number of selection systems can be used. For example, the herpessimplex virus thymidine kinase (see, e.g., Wigler et al., 1977, Cell11:223), hypoxanthine-guanine phosphoribosyltransferase (see, e.g.,Szybalska and Szybalski, 1992, Proc. Natl Acad. Sci. USA 48:202), andadenine phosphoribosyltransferase (see, e.g., Lowy et al., 1980, Cell22:817) genes can be employed in tk-, hgprt- or aprt-cells,respectively. Also, antimetabolite resistance can be used as the basisof selection for the following genes: DHFR, which confers resistance tomethotrexate (see e.g., Wigler et al., 1980, Proc. Natl. Acad Sci. USA77:3567-70; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527-31); gpt, which confers resistance to mycophenolic acid (see, e.g.,Mulligan and Berg, 1981, Proc. Nail. Acad. Sci. USA 78:2072-76); neo,which confers resistance to the aminoglycoside G-418 (see, e.g.,Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95;Tolstoshev, 1993, Arm. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan,1993, Science 260:926-932; Morgan and Anderson, 1993, Ann. Rev. Biochem.62:191-217; and May, 1993, TIB TECH 11(5): 155-215) and hygro, whichconfers resistance to hygromycin (see, e.g., Santerre et al., 1984, Gene30:147-50). Methods commonly known in the art of recombinant DNAtechnology which can be used are described in Ausubel et al. (eds.,1993-2006, Current Protocols in Molecular Biology. John Wiley & Sons,NY; Kriegler, 1990, Gene Transfer and Expression. A laboratory Manual,Stockton Press, NY; and in Chapters 12 and 13, Dracopoli et al. (eds.,1994, Current Protocols in Human Genetics, John Wiley & Sons, NY; andColberre-Garapin et al, 1981, 7. MoI. Biol. 150:1-14).

The expression levels of an antibody can be increased by vectoramplification (for a review, see Bebbington and Hentschel, The use ofvectors based on gene amplification for the expression of cloned genesin mammalian cells in DNA cloning, Vol. 3. (Academic Press, New York,1987)). When a marker in the vector system expressing an antibody isamplifiable, an increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the nucleotide sequence of theantibody, production of the antibody will also increase (see, e.g.,Crouse et al., 1983, Mol. Cell. Biol. 3:257-66).

The host cell can be co-transfected with two expression vectors, thefirst vector encoding a heavy chain derived polypeptide and the secondvector encoding a light chain derived polypeptide. The two vectors cancontain the same or different selectable markers that enable equalexpression of heavy and light chain polypeptides. Alternatively, asingle vector can be used to encode both heavy and light chainpolypeptides. In such situations, the light chain is typically placedbefore the heavy chain to avoid an excess of toxic free heavy chain(see, e.g., Proudfoot, 1986, Nature 322:562-65; Kohler, 1980, Proc.Natl. Acad. Sci. USA 77:2197-9). The coding sequences for the heavy andlight chains can comprise cDNA or genomic DNA.

Once the antibody has been recombinantly expressed, it can be purifiedusing any suitable method for purification of an antibody, for example,by chromatography (e.g., ion exchange, affinity, particularly byaffinity for the specific antigen after Protein A, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins.

A comprehensive reference for all steps used for making the monoclonalantibodies according to the present invention is Yokoyama et al.,“Production of Monoclonal Antibodies”, Current Protocols in Immunology,Unit 2.5, 2006.

EXAMPLES Example 1 Affinity to CD33

The CD33 binding agents have a KD to both human and cynomolgus CD33 ofAffinities of 10 nM or less on the cell line HL60 and HEK293-cynomolgusCD33, respectively.

Fourteen CD33 binding agents (fully human monoclonal antibodies aslisted in table 1 as No. 1-14, respectively) to human and cynomolgusCD33 were determined by FACS Scatchard analysis as described inBrockhoff et al., (Cytometry. 1994 Sep. 1; 17(1):75-83) on CD33expressing cells (AML-derived HL60 cell line, recombinantHEK293-cynomolgus CD33 cell line). Briefly, dilutions of a CD33 bindingagent were prepared in a 96 well plate starting with 100-400 nM in thefirst well (80 μl), followed by 11 dilution steps (1:2, 40+40 μl). 50 μlof CD33 binding agent dilutions are added to FACS tubes, 150 μl cells(0.8×10⁶/ml=1.2×10⁵ cells/tube) are added to each FACS tube. Cells weregently mixed and incubated for 1 h on ice. Thereafter 50 μl FITCconjugated secondary antibody (concentration 15 μg/ml; mouse mAbanti-human IgG) was added, mixed, and incubated for 30 min on ice. 4 mlPBS ph7.2 containing 0.02% acid were added thereafter, cells werepelleted and resuspended in 300 μl PBS pH 7.2 and subjected to FACSanalysis using a BD FACS Canto. All experimental steps were performed onwet ice, all CD33 binding agent dilutions were made in PBS/0.5%BSA+0.02% acid. FACS calibration was performed using Quantum FITC MESF(Premix) Beads (Bangs Laboratories). All samples were measured using thesame FACS parameters. The ratio of bound IgG versus free IgG wascalculated from MFI values at different CD33 binding agentconcentrations and displayed as Scatchard blot. A regression line wasdrawn through the resulting data points, the slope of this linecorresponds to the negative value of the association constant. Theresults are listed in table 2.

TABLE 2 Human CD33 Cynomolgus CD33 No Clone ID # K_(D) (nM) K_(D) (nM) 1280-03-08 0.3 0.03 2 280-21-09 0.4 0.3 3 280-29-12 0.5 0.6 4 280-31-010.4 0.3 5 280-31-01(mut) 1 0.5 6 280-34-02 0.3 0.5 7 280-50-01 0.3 0.5 8280-50-01(mut) 0.4 1.7 9 280-61-07 0.3 0.4 10 283-03-03 0.3 1.9 11283-05-01 0.2 1.1 12 283-07-03 0.3 2.1 13 283-11-03 0.3 0.6 14 283-14-013.2 2.3

Example 2 Internalization Kinetics

Internalization of antibody refers to the reduction of the amount ofantibody/antigen complexes on the cell surface of the target cell afterincubation with antibody. Internalization tests were performed with theCD33 expressing HL60 cell line. Cells were incubated with a fixed amountof a CD33 binding agent (10 μg/ml of fully human monoclonal antibodiesas listed in table 1 as No. 1-14) for defined time periods (0 h, 1 h, 4h, 24 h) at 37° C. to allow internalization of the antibody/antigencomplex. At the indicated time points acid was added to the incubationmix to prevent further internalization. Thereafter a fixed amount ofCD33 binding agent was added to saturate all CD33 antigen sites on thecell surface. The total amount of CD33 binding agent bound to the cellsurface was determined by FACS analysis utilizing a FITC conjugatedanti-human IgG secondary antibody. The time point 0 h was used todetermine the initial level of CD33 antibody/antigen complexes on thesurface and was defined as 100%. The results are listed in table 3 andillustrated in FIGS. 1-3.

TABLE 3 Remaining CD33 antibody/antigen complexes on the cells surfaceafter 4 h No Clone ID # incubation with antibody (%) 1 280-03-08 49 2280-21-09 41 3 280-29-12 49 4 280-31-01 44 5 280-31-01(mut) 52 6280-34-02 44 7 280-50-01 53 8 280-50-01(mut) 55 9 280-61-07 47 10283-03-03 45 11 283-05-01 51 12 283-07-03 48 13 283-11-03 46 14283-14-01 47

Lintuzumab was included as a reference antibody. Lintuzumab/CD33complexes rapidly internalized upon binding of lintuzumab which was inaccordance to published data. After a 4 h-incubation period only about20% of the initial amount of CD33/lintuzumab complexes was left on thecell surface. It could be demonstrated that, unexpectedly,internalization of all 14 CD33 binding agents according to the presentinvention was decelerated compared to lintuzumab.

Example 3 ADCC Activity

Decelerated internalization rate translates into increased ADCC activityin vitro. To assess the effect of decelerated internalization on theADCC activity of the CD33 binding agents (fully human monoclonalantibodies as listed in table 1 as No. 1-14), target cells (HL60) wereincubated with a CD33 binding agent for the 0, 1, 4 and 24 h.Subsequently an ADCC assay was performed with IL-2 stimulated PBMC aseffector cells and antibody coated HL60 cells as target cells. For allexperiments a mAb concentration of 30 μg/ml used. The co-cultivation ofeffector cells with target cells in presence of CD33 binding agent wasperformed in quadruplicates or triplicates in 96-well round-bottommicrotiter plates in a final volume of 200 μl assay medium per wellconsisting of 10% human serum and 1% BSA in RPMI in 1:1 ratio. Firsteffector cells (freshly isolated PBMC cells in 100 μl 10% human serum inRPMI per well) were plated, followed by target cells and CD33 bindingagent solution (diluted in 50 μl 1% BSA in RPMI). As a control, effectorcells were cultivated in assay medium alone (effector cell control) andtarget cells were cultivated either in assay medium alone (spontaneouslysis) or in assay medium supplemented with 1% Triton X-100 (maximallysis). The co-culture was incubated at 37° C. in a humid CO₂ incubatorfor 3 hours. At the end of the incubation cells were removed from theculture medium by centrifugation (200×g, i.e. 1000 rpm; 10 min) at roomtemperature. Cell free supernatants (100 μl/well) are transferred intocorresponding wells of a 96-well flat-bottom plate. To determine the LDHactivity in these supernatants 100 μl reaction mixture (freshly mixed250 μl catalyst with 11.25 ml dye solution) are added to each well andincubated 30 min at room temperature in the dark. Then the absorbance ismeasured as described below.

Cytotoxicity Detection Kit (LDH; Roche 11 644 793 001) was used tomeasure ADCC activity. The detection of cytotoxicity is based on themeasurement of LDH enzyme activity released from plasma membrane-damagedcells. LDH released into the culture supernatants reduces thetetrazolium salt from the kit to formazan. The absorption maximum offormazan dye is measured at 490 nm against a reference wavelength of 650nm in an ELISA plate reader. To determine the percentage cell mediatedcytotoxicity the average absorbance of the quadruplicates or triplicateswas calculated and the background was subtracted. These corrected valueswere substituted into the following equation to calculate ADCC (%):(effector/target cell mix-effector cell control-spontaneous release)divided by (maximal release-spontaneous release)

ADCC activity at timepoint 0 h (no antibody pre-incubation of targetcells) was defined as 100% ADCC activity. ADCC activity for varioustimepoints of antibody pre-incubation was calculated relative totimepoint 0 h and displayed as relative cytotoxicity (%).

Decelerated internalization of the CD33 binding agents compared tolintuzumab resulted in increased ADCC activity compared to lintuzumab.In conclusion, decelerated internalization leads to increased ADCCactivity. Internalization for the described CD33 binding agents isinversely correlated to ADCC activity of the CD33 binding agents whichis indicative for advantages with respect to clinical activity of suchCD33 binding agents. The results are illustrated in FIG. 4 for theinternalization kinetics in this experiment and in FIG. 5 for the ADCCactivity.

Example 4 Epitope Mapping

The binding epitope of CD33 binding agents described herein relative tothe epitope of lintuzumab was determined by Hydrogen Exchange MassSpectrometry (HXMS).

This method determined the susceptibility of the amide backbonehydrogens of CD33 protein to exchange with D₂O. The experiments wereconducted with recombinant CD33 protein alone and CD33 protein withadded CD33 binding agent/lintuzumab (in the following in this examplereferred to as “antibody/antibodies”). Regions of the CD33 proteinshowing significant protection from exchange due to binding of antibodywere thus identified. Resolution of the method is determined by thepeptides produced by digestion with pepsin, e.g. the resulting aminoacid sequence may be larger than the actual epitope of the antibody.These CD33 derived peptides were identified by additional controlexperiments with unexchanged samples employing standard accurate massand HPLC MS/MS technologies.

For the protein+antibody sample, CD33 protein and antibody wereincubated for 15 minutes at room temperature. The final molar ratioantibody/CD33 was 2:1. Using a LEAP robot system (exchange plate kept at25° C., sample/quench plate kept at 4° C.), 8 ul of sample was added to80 μl of exchange buffer (10 mM NaH₂PO₄ in D₂O, pH=7 or 10 mM NaH₂PO₄ inH₂O, pH=7), mixed, and allowed to exchange for various times (15, 60,120, 240, and 600 seconds) at 25° C. 80u1 of this solution was thentransferred to 80 μl of quench buffer (1M urea, 0.1M TCEP-HCl) at 4° C.and mixed. 90 μl of this solution was then transferred to 10 μl ofpepsin (4 mg/me at 4° C. and mixed. After 2 minutes, 60 μl of thissolution was injected onto a Michrom C18 trap cartridge. The cartridgewas washed with H₂O+0.1% TFA for 2 minutes at 100 μl/min A valve wasthen switched and the cartridge eluted onto a Phenomenex Jupiter C5column, 1.0×5 mm, 5 μm, 300 A. Mobile Phase A was water/acetonitrile/TFA(99/0.95/0.05) and Mobile Phase B was acetonitrile/water/TFA(95/4.95/0.05). Flow rate was 100 μl/min. Gradient was: 0 minutes (0%B), 6 minutes (40% B), 7 minutes (40% B), 8 minutes (90% B), 10 minutes(90% B), 11 minutes (0% B). The LEAP system precools the mobile phase to4° C. and also maintains the trap column and analytical column at 4° C.For the MS experiments (used to quantitate exchange with the D₂Obuffer), a single scan method from 300-2000 for 14 minutes was used atresolution 60,000. For the MS/MS experiments (used to ID peptides withthe H₂O exchange buffer), a method with 7 scans was used for 14 minutes.The first scan was a full range scan from 300-2000 at 30,000 resolution.Subsequent scans were CID scans of the 6 most intense ions from scan #1.Isolation width was 1.5 amu, collision energy was 35V, activation timewas 3 msec. Pepsin peptides were identified using fragmentation data andthe program Proteome Discoverer (Thermo). Identified peptides wereanalyzed using an in-house program which calculates the average mass forexchanged peptides.

All CD33 binding agents protected the identical peptide fragment withthe amino acid sequence FFHPIPYYDKNSPVHGYW (Seq ID No: 141) (Table 4).The sequence of CD33 protected by the CD33 binding agents describedherein are different and non-overlapping to the peptide sequence of CD33protected by binding of Lintuzumab (MDPNFWLQVQE, Seq ID No: 142). Insilico modeling using the crystal structure of SIGLEC-5, a SIGLEC familymember homologous to CD33, revealed binding epitopes of all antibodiesin the proximal domain of the protein, where the binding epitope ofLintuzumab is different to those of the CD33 binding agents describedherein. In conclusion, the CD33 binding agents described in this patentapplication bind to a different epitope than Lintuzumab.

TABLE 4 No Clone ID # CD33 Epitope  1 280-03-08 FFHPIPYYDKNSPVHGYW  2280-21-09 FFHPIPYYDKNSPVHGYW  3 280-29-12 FFHPIPYYDKNSPVHGYW  4280-31-01 FFHPIPYYDKNSPVHGYW  5 280-31-01 (mut) FFHPIPYYDKNSPVHGYW  6280-34-02 FFHPIPYYDKNSPVHGYW  7 280-50-01 FFHPIPYYDKNSPVHGYW  8280-50-01 (mut) FFHPIPYYDKNSPVHGYW  9 280-61-07 FFHPIPYYDKNSPVHGYW 10283-03-03 FFHPIPYYDKNSPVHGYW 11 283-05-01 FFHPIPYYDKNSPVHGYW 12283-07-03 FFHPIPYYDKNSPVHGYW 13 283-11-03 FFHPIPYYDKNSPVHGYW 14283-14-01 FFHPIPYYDKNSPVHGYW 15 Lintuzumab MDPNFWLQVQE

1. A CD33 binding agent binding to human CD33, the CD33 binding agentbeing defined by a) having a heavy chain variable region comprisingCDR1, CDR2 and CDR3, and a light chain variable region comprising CDR4,CDR5 and CDR6, wherein CDR1 has an amino acid sequence selected fromSeqID No:1-14, CDR2 has an amino acid sequence selected from SeqIDNo:15-28, CDR3 has an amino acid sequence selected from SeqID No:29-42,CDR4 has an amino acid sequence selected from SeqID No: 43-56, CDR5 hasan amino acid sequence selected from SeqID No: 57-70, CDR6 has an aminoacid sequence selected from SeqID No: 71-84, or b) recognizing anepitope within the amino acid sequence FFHPIPYYDKNSPVHGYW (SeqID No:141) of human CD33.
 2. The CD33 binding agent according to claim 1,wherein the epitope is determined by Hydrogen Exchange MassSpectroscopy.
 3. The CD33 binding agent according to claim 1, whereinthe internalization kinetics of the CD33 binding agent are such that atleast 30% of the initial amount of the CD33 binding agent remains on thecell surface of HL60 cells at a time of 4 hours after incubation.
 4. TheCD33 binding agent according to claim 3, wherein the internalizationkinetics of the CD33 binding agents are such that at least 40% of theinitial amount of the CD33 binding agents remain on the cell surface ata time of 4 hours after incubation.
 5. The CD33 binding agent accordingto claim 1, wherein the heavy chain variable region comprises an aminoacid sequence selected from SeqID No:85-98 and the light chain variableregion comprises an amino acid sequence selected from SeqID No:99-112.6. The CD33 binding agent according to claim 1, wherein the CD33 bindingagent has a heavy chain having an amino acid sequence selected fromSeqID No: 113-126 and a light chain having an amino acid sequenceselected from SeqID No: 127-140.
 7. The CD33 binding agent according toclaim 1, wherein the CD33 binding agent has an affinity to both humanCD33 and cynomolgus CD33 with a KD of equal to or less than 10 nM. 8.The CD33 binding agent according to claim 1, wherein the CD33 bindingagent is humanized
 9. The CD33 binding agent according to claim 1,wherein the CD33 binding agent is fully human.
 10. The CD33 bindingagent according to claim 1, wherein the CD33 binding agent furthercomprises an effector function.
 11. The CD33 binding agent according toclaim 10, wherein the effector function is mediated by an F_(c) domain.12. The CD33 binding agent according to claim 10, wherein the CD33binding agent comprises one or more mutations in the F_(c) domain thatmodulate the function of the F_(c) domain.
 13. The CD33 binding agentaccording to claim 12, wherein the modulation of the function of the Fcdomain is an increase of ADCC by at least 10%, by at least 50% or by atleast 100%.
 14. The CD33 binding agent according to claim 12, whereinthe one or more mutations in the F_(c) domain are located at one or morepositions selected from amino acids at positions 332 and/or 239 and/or236 according to the Kabat EU numbering index.
 15. The CD33 bindingagent according to claim 12, wherein the one or more mutations in theF_(c) domain are a combination of substitutions at positions 239 and332, preferably S239D/I332E.
 16. The CD33 binding agent according toclaim 15, wherein the mutations in the F_(c) domain are a combination ofsubstitutions at S239D/I332E.
 17. A CD33 binding agent selected from anantibody comprising a CDR1 of SeqID No: 1, a CDR2 of SeqID No: 15, aCDR3 of SeqID No: 29, a CDR4 of SeqID No: 43, a CDR5 of SeqID No: 57 anda CDR6 of SeqID No: 71, an antibody comprising a CDR1 of SeqID No: 2, aCDR2 of SeqID No: 16, a CDR3 of SeqID No: 30, a CDR4 of SeqID No: 44, aCDR5 of SeqID No: 58 and a CDR6 of SeqID No: 72, an antibody comprisinga CDR1 of SeqID No: 3, a CDR2 of SeqID No: 17, a CDR3 of SeqID No: 31, aCDR4 of SeqID No: 45, a CDR5 of SeqID No: 59 and a CDR6 of SeqID No: 73,an antibody comprising a CDR1 of SeqID No: 4, a CDR2 of SeqID No: 18, aCDR3 of SeqID No: 32, a CDR4 of SeqID No: 46, a CDR5 of SeqID No: 60 anda CDR6 of SeqID No: 74, an antibody comprising a CDR1 of SeqID No: 5, aCDR2 of SeqID No: 19, a CDR3 of SeqID No: 33, a CDR4 of SeqID No: 47, aCDR5 of SeqID No: 61 and a CDR6 of SeqID No: 75, an antibody comprisinga CDR1 of SeqID No: 6, a CDR2 of SeqID No: 20, a CDR3 of SeqID No: 34, aCDR4 of SeqID No: 48, a CDR5 of SeqID No: 62 and a CDR6 of SeqID No: 76,an antibody comprising a CDR1 of SeqID No: 7, a CDR2 of SeqID No: 21, aCDR3 of SeqID No: 35, a CDR4 of SeqID No: 49, a CDR5 of SeqID No: 63 anda CDR6 of SeqID No: 77, an antibody comprising a CDR1 of SeqID No: 8, aCDR2 of SeqID No: 22, a CDR3 of SeqID No: 36, a CDR4 of SeqID No: 50, aCDR5 of SeqID No: 64 and a CDR6 of SeqID No: 78, an antibody comprisinga CDR1 of SeqID No: 9, a CDR2 of SeqID No: 23, a CDR3 of SeqID No: 37, aCDR4 of SeqID No: 51, a CDR5 of SeqID No: 65 and a CDR6 of SeqID No: 79,an antibody comprising a CDR1 of SeqID No: 10, a CDR2 of SeqID No: 24, aCDR3 of SeqID No: 38, a CDR4 of SeqID No: 52, a CDR5 of SeqID No: 66 anda CDR6 of SeqID No: 80, an antibody comprising a CDR1 of SeqID No: 11, aCDR2 of SeqID No: 25, a CDR3 of SeqID No: 39, a CDR4 of SeqID No: 53, aCDR5 of SeqID No: 67 and a CDR6 of SeqID No: 81, an antibody comprisinga CDR1 of SeqID No: 12, a CDR2 of SeqID No: 26, a CDR3 of SeqID No: 40,a CDR4 of SeqID No: 54, a CDR5 of SeqID No: 68 and a CDR6 of SeqID No:82, an antibody comprising a CDR1 of SeqID No: 13, a CDR2 of SeqID No:27, a CDR3 of SeqID No: 41, a CDR4 of SeqID No: 55, a CDR5 of SeqID No:69 and a CDR6 of SeqID No: 83, or an antibody comprising a CDR1 of SeqIDNo: 14, a CDR2 of SeqID No: 28, a CDR3 of SeqID No: 42, a CDR4 of SeqIDNo: 56, a CDR5 of SeqID No: 70 and a CDR6 of SeqID No:
 84. 18. A CD33binding agent selected from an antibody comprising a heavy chainvariable region of SeqID No: 85 and a light chain variable region ofSeqID No: 99, an antibody comprising a heavy chain variable region ofSeqID No: 86 and a light chain variable region of SeqID No: 100, anantibody comprising a heavy chain variable region of SeqID No: 87 and alight chain variable region of SeqID No: 101, an antibody comprising aheavy chain variable region of SeqID No: 88 and a light chain variableregion of SeqID No: 102, an antibody comprising a heavy chain variableregion of SeqID No: 89 and a light chain variable region of SeqID No:103, an antibody comprising a heavy chain variable region of SeqID No:90 and a light chain variable region of SeqID No: 104, an antibodycomprising a heavy chain variable region of SeqID No: 91 and a lightchain variable region of SeqID No: 105, an antibody comprising a heavychain variable region of SeqID No: 92 and a light chain variable regionof SeqID No: 106, an antibody comprising a heavy chain variable regionof SeqID No: 93 and a light chain variable region of SeqID No: 107, anantibody comprising a heavy chain variable region of SeqID No: 94 and alight chain variable region of SeqID No: 108, an antibody comprising aheavy chain variable region of SeqID No: 95 and a light chain variableregion of SeqID No: 109, an antibody comprising a heavy chain variableregion of SeqID No: 96 and a light chain variable region of SeqID No:110, an antibody comprising a heavy chain variable region of SeqID No:97 and a light chain variable region of SeqID No: 111, or an antibodycomprising a heavy chain variable region of SeqID No: 98 and a lightchain variable region of SeqID No:
 113. 19. A CD33 binding agentselected from an antibody comprising a heavy chain of SeqID No: 113 anda light chain of SeqID No: 127, an antibody comprising a heavy chain ofSeqID No: 114 and a light chain of SeqID No: 128, an antibody comprisinga heavy chain of SeqID No: 115 and a light chain of SeqID No: 129, anantibody comprising a heavy chain of SeqID No: 116 and a light chain ofSeqID No: 130, an antibody comprising a heavy chain of SeqID No: 117 anda light chain of SeqID No: 131, an antibody comprising a heavy chain ofSeqID No: 118 and a light chain of SeqID No: 132, an antibody comprisinga heavy chain of SeqID No: 119 and a light chain of SeqID No: 133, anantibody comprising a heavy chain of SeqID No: 120 and a light chain ofSeqID No: 134, an antibody comprising a heavy chain of SeqID No: 121 anda light chain of SeqID No: 135, an antibody comprising a heavy chain ofSeqID No: 122 and a light chain of SeqID No: 136, an antibody comprisinga heavy chain of SeqID No: 123 and a light chain of SeqID No: 137, anantibody comprising a heavy chain of SeqID No: 124 and a light chain ofSeqID No: 138, an antibody comprising a heavy chain of SeqID No: 125 anda light chain of SeqID No: 139, or an antibody comprising a heavy chainof SeqID No: 126 and a light chain of SeqID No:
 140. 20. A DNA moleculecomprising a region encoding the heavy chain variable region or a lightchain variable region of a CD33 binding agent according to claim 1.21-30. (canceled)